Name: a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model
Uploaded: 2016-10-14T14:00:00.000+00:00
Duration: 8 min
Description: Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

The authors would like to acknowledge the "Ligue Nationale contre le Cancer" (Comit&#233; du Septentrion), the SIRIC ONCOLille (Grant INCa-DGOS-INSERM 6041) and the Institut pour la Recherche sur le Cancer de Lille (IRCL) for supporting this work. They would like to thank Delphine Taillieu and the animal facility staff for housing the mice and maintaining their welfare. We also thank Rapha&#235;lle Caillerez and Nathalie Jouy for their respective help in microscopy and flow cytometry.

<ol>
	<li>Forthun, R. B., Hinrichs, C., Dowling, T. H., Bruserud, &. #. 2. 1. 6. ;., Selheim, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+past,+present+and+future+subclassification+of+patients+with+acute+myeloid+leukemia.">The past, present and future subclassification of patients with acute myeloid leukemia.</a> Curr. Pharm. Biotechnol. 17 (1), 6-19 (2016).</li><li>Goldie, H., Butler, C. H., Anderson, M. M., Maxwell, M. C., Hahn, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+characteristics+of+free+C1498+(granulocytic+leukemia)+tumor+cells+in+the+peritoneal+fluid+and+the+blood+of+C57+mice.">Growth characteristics of free C1498 (granulocytic leukemia) tumor cells in the peritoneal fluid and the blood of C57 mice.</a> Cancer Res. 13 (2), 125-129 (1953).</li><li>Tanaka, K. K., Roberts, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+studies+of+E.L.4+lymphoma+and+C+Leukemia+in+susceptible+(C57BL)+and+resistant+(B10.D2)+mice.">Biological studies of E.L.4 lymphoma and C Leukemia in susceptible (C57BL) and resistant (B10.D2) mice.</a> Cancer Res. 24 (1498), 1785-1797 (1964).</li><li>Law, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+influence+affecting+growth+of+transplantable+leukemias+in+mice.">Characterization of an influence affecting growth of transplantable leukemias in mice.</a> Cancer Res. 4, 257-260 (1944).</li><li>Graham, J. D., McMahon Welch, C., Patchen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+of+an+implanted+murine+myelogenous+leukemia+C1498.">Studies of an implanted murine myelogenous leukemia C1498.</a> Ohio J. Sci. 75 (4), 202-208 (1975).</li><li>Boyer, M. W., Orchard, P. J., Gorden, K. B., Anderson, P. M., Mclvor, R. S., Blazar, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependency+on+intercellular+adhesion+molecule+recognition+and+local+interleukin-2+provision+in+generation+of+an+in+vivo+CD8++T-cell+immune+response+to+murine+myeloid+leukemia.">Dependency on intercellular adhesion molecule recognition and local interleukin-2 provision in generation of an in vivo CD8+ T-cell immune response to murine myeloid leukemia.</a> Blood. 85 (9), 2498-2506 (1995).</li><li>Labelle, J. L., Truitt, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+murine+NKT+cell+tumor+previously+described+as+an+acute+myelogenous+leukemia.">Characterization of a murine NKT cell tumor previously described as an acute myelogenous leukemia.</a> Leuk. Lymphoma. 43 (8), 1637-1644 (2002).</li><li>Bradner, W. T., Pindell, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myeloid+leukemia+as+a+screen+for+cancer+chemotherapeutic+agents.">Myeloid leukemia as a screen for cancer chemotherapeutic agents.</a> Cancer Res. 26 (4), 375-390 (1966).</li><li>Lin, J. M., Li, B., Rimmer, E., VanRoey, M., Jooss, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+the+anti-tumor+efficacy+of+a+GM-CSF-secreting+tumor+cell+immunotherapy+in+preclinical+models+by+cytosine+arabinoside.">Enhancement of the anti-tumor efficacy of a GM-CSF-secreting tumor cell immunotherapy in preclinical models by cytosine arabinoside.</a> Exp. Hematol. 36 (3), 319-329 (2008).</li><li>Robinson, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Flow Cytometry Methods. , (1993).</li><li>Xu, Y., McKenna, R. W., Wilson, K. S., Karandikar, N. J., Schultz, R. A., Kroft, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotypic+identification+of+acute+myeloid+leukemia+with+monocytic+differentiation.">Immunophenotypic identification of acute myeloid leukemia with monocytic differentiation.</a> Leukemia. 20 (7), 1321-1324 (2006).</li><li>Hassan, K., Qureshi, M., Shafi, S., Ikram, N., Akhtar, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myeloid+leukemia-FAB+classification+and+its+correlation+with+clinico-haematological+features.">Acute myeloid leukemia-FAB classification and its correlation with clinico-haematological features.</a> J. Pak. Med. Assoc. 43 (10), 200-203 (1993).</li><li>Quesenberry, P. J., Rappeport, J. M., Fountebouni, A., Sullivan, R., Zuckerman, K., Ryan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+normal+murine+hematopoiesis+by+leukemic+cells.">Inhibition of normal murine hematopoiesis by leukemic cells.</a> N.Engl.J. Med. 299 (2), 71-75 (1978).</li><li>Youn, J. I., Nagaraj, S., Collazo, M., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets+of+myeloid-derived+suppressor+cells+in+tumor-bearing+mice.">Subsets of myeloid-derived suppressor cells in tumor-bearing mice.</a> J. Immunol. 181 (8), 5791-5802 (2008).</li><li>Loder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+development+in+the+spleen+takes+place+in+discrete+steps+and+is+determined+by+the+quality+of+B+cell+receptor-derived+signals.">B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals.</a> J. Exp. Med. 190 (1), 75-89 (1999).</li><li>Robbins, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extramedullary+hematopoiesis+generates+Ly-6C(high)+monocytes+that+infiltrate+atherosclerotic+lesions.">Extramedullary hematopoiesis generates Ly-6C(high) monocytes that infiltrate atherosclerotic lesions.</a> Circulation. 125 (2), 364-374 (2012).</li></ol>

The authors declare that they have no competing financial interests.

In früheren Studien wurde die C1498 - Zelllinie als Induktor von akuter granulozytärer 5 beschriebenen myelomonocytic 6 oder 7 NKT - Zell - Leukämie. Allerdings demonstrative Daten in der Literatur waren entweder nicht vorhanden oder unvollständig sind. Das Protokoll vorgestellt hier verwendet verschiedene Techniken, wie Durchflusszytometrie, Immunofluoreszenz, MGG-Färbung und cytochemischen Assays kultiviert C1498-Zellen zu charakterisieren und die Art der Leukämie zu bestimmen, die in Mäusen induziert wird, nachdem sie injiziert werden. Wenn wir in vitro kultiviert C1498 - Zellen nach Immunfärbung phänotypisiert und Zytometrie Analysen Fluss durchgeführt wurden, beobachteten wir einige Einschränkungen , da diese Zellen einige Zelloberfläche hämatopoetischen Marker exprimiert , die 6,7 in der Literatur bereits beschrieben worden sind. In Übereinstimmung mit unseren Ergebnissen Labelle et al. Haben die Zelloberflächenexpression von reifen TCR auf C1498 - Zellen unter Verwendung von Fluss cytomet nicht beobachtenry Färbung. Allerdings hielten sie ihnen eine NKT - Zelllinie zu sein , nachdem sie 7 CD3 &amp; egr; und TCRVβ8.2 mRNAs nachgewiesen. Wir beobachteten auch die intrazelluläre Expression von CD3 &amp; egr; TCRVβ Ketten und Moleküle in die meisten Zellen (&gt; 70%), aber ihre hämatopoetischen Abstammungslinien konnte nicht bestimmt werden, weil es auch die gleichzeitige intrazelluläre Expression des Moleküls Mac-3 war. Myeloperoxidase, MGG-Färbung und Einschätzungen funktionelle Esterasen zu analysieren Zytochemie mit gezeigt, dass die C1498-Zelllinie eine myeloische Ursprung hatte und von Monoblasten und Myeloblasten zusammen. Diese Ergebnisse waren konkordant mit dem Prozentsatz der Mac-3 + Zellen , die in Durchflusszytometrie Färbungsanalyse erhalten wurden. Obwohl nicht quantitative, diese Schritte Schlüsselexperimente darstellen durchgeführt werden. Tatsächlich bleiben sie bisher die besten bestehenden Methoden zur Charakterisierung der Linie und Differenzierungsstadium von hämatopoetischen Zellen, die keine oder f ausdrückenew spezifische phänotypische Marker. Die Durchflusszytometrie Färbung war hilfreich für die Entwicklung von akuter Leukämie in congenic Mäusen demonstriert nach C1498-Zellen wurden intravenös injiziert. Die CD45.2 + C1498 - Zellen , die in das periphere Blut und verschiedene Organe infiltriert wurden isoliert, und ihre Frequenzen bestimmt. Eine quantitative Bestimmung wurde auch inhärente Mark und Milz-Zellen nach Immunophänotypisierung durchgeführt, um zu analysieren. Einschränkungen aufgetreten sind , wenn die C1498 Zellphänotyps in Organen wurde untersucht , wie sie einige hämatopoetischen Marker exprimiert (nur einige von ihnen waren B220 +). So definieren die Art der beobachteten akuten Leukämie, May-Grünwald-Giemsa-Färbung und eine Analyse der Aktivitäten von monozytären und granulocytic Esterasen wurden unter Verwendung von Knochenmark durchgeführt. Die Ergebnisse zeigten, dass C1498 Zellen behielten ihre myeloische und monoblastischen Morphologie und Funktion, den Beginn der myelomonocytic Leukämie enthüllt. <p class = &quot;jove_content&quot;&gt; Als Gegenleistung für die kritischen Schritte in diesem Protokoll beschrieben ist, sollte besonderes Augenmerk auf pH-Wert gegeben werden, wenn cytochemischer Reaktionen und MGG-Färbung durchzuführen, da Fehler in pH-Wert zu Fehlinterpretationen der Ergebnisse führen kann. Beispielsweise Esteraseaktivität α-naphthyl Butyrat ist spezifisch für Monozyten-Zellen, die nur bei einem pH von 6,0, da Granulozyten und Lymphozyten auch positiv für diesen Test bei höheren pH-Werten beflecken kann. die Zellen fixiert wird nicht vor der Durchführung MGG-Färbung empfohlen, und wir haben gezeigt, dass nur CAF Fixierung zufriedenstellende Ergebnisse zur Verfügung gestellt, wenn Esterasen cytochemischer Reaktionen unter Verwendung von C1498-Zellen durchführen. Zu erhalten Zytometrie die Expression des CD115 - Moleküls und dessen Detektion durch Strömungs, alle Proben (beispielsweise Blut, Knochenmark und Milzzellen) sollten auf Eis während des Verfahrens gehalten werden. Wenn keine Färbung wird in der Durchflusszytometrie oder / und Immunofluoreszenz Experimente, die Referenz der Antikörper, deren Lagerung re beobachtetLob und ihre Verdünnungen sollten überprüft werden. Die Verweise in den Materialien / Ausrüstung Tabelle angegeben wurden für die Durchflusszytometrie oder Immunofluoreszenz Anwendungen ausgewählt. Die primäre / sekundäre Antikörper oder deren konjugierte Fluorophore könnten ihre Aktivität durch falsche Lagerung verloren haben (zB Einwirkung von Licht oder Wärme), unsachgemäße Verdünnung, umfangreiche Einfrieren / Auftauen oder die Verwendung von kontaminierten Puffer. Führen positive Kontrollen, um sicherzustellen, dass sie ordnungsgemäß funktionieren. Mit der Maus Knochenmark oder Milz-abgeleiteten Zellen, die die Proteine ​​von Interesse sind dafür bekannt, zum Ausdruck bringen. Um eine hohe Hintergrund und nicht-spezifische Färbung zu vermeiden, stellen Sie sicher, dass sich die Zellen richtig gewaschen werden und gehalten bei hoher Luftfeuchtigkeit (für Immunofluoreszenz) und dass die Antikörper verdünnt, wie angewiesen. Verwenden Sie die gleiche Konzentration und Verdünnung für die Isotypkontrollantikörper und dem primären Antikörper, um genau die Hintergrundebene in der Probe zu bestimmen. Für Esterase Zytochemie Experimenten wurde dieReagenzien können mit positiven und negativen Steuerschiebern enthalten , gereinigter Maus splenic granulocytic (Ly6G +) und monozytären (CD115 + Zellen) getestet werden. Das Verfahren in dieser Studie beschrieben , zeigte , dass viele der leukämischen Merkmale in Mäusen nach der Injektion von C1498 - Zellen beobachtet geteilt gemeinsame Merkmale mit der menschlichen akuten myelomonozytäre Leukämie 11,12. Die Invasion leukämischen Zellen führte zu einer Reduktion von reifen und unreifen (Vorläufern und Vorprodukte) Mark hämatopoetischen Zellen. C1498-Zellen vorhanden sind, mit hoher Frequenz (&gt; 20%) in dem peripheren Blut, wie monozytären Zellen sind. Hepatomegalie und Splenomegalie beobachtet von der Infiltration von leukämischen Zellen führen und deutliche Steigerungen in B-Lymphozyten und myeloischen Zellen wurden auch Splenomegalie zu begleiten beobachtet. Thrombopenie wurde auch beobachtet, wenn Blutplättchen Zahlen geschätzt wurden ein Hämatologie-Analysator. Es war Shown, in - vitro - Experimenten mit, dass C1498 Zellen hemmen normalen murinen Hämatopoese durch lösliche Faktoren 13 sezer. In mehreren Tumormausmodellen, unreifen myeloischen Zellen (einschließlich Monozyten - und granulozytäre Zellen) haben auch gezeigt, aus dem Knochenmark in die Milz zu wandern, wo sie 14 anti-Tumor - spezifische T - Zell - Aktivierung und Proliferation hemmen. So haben die Reduktion in hämatopoetischen Zellen, die im Knochenmark beobachtet wurde, könnte entweder von einem Mangel in der Hämatopoese und / oder von ihrer Auswanderung geführt. Dieser letztere Mechanismus könnte die Anwesenheit von Monozytose im peripheren Blut oder die Beobachtung der vergrößerten myeloische Fraktionen in der Milz erklären. Es ist auch denkbar, dass diese Zellen von einem verbesserten splenic Hämatopoese abgeleitet worden sein könnte. Tatsächlich unter stationären Bedingungen wurden einige Untergruppen von Milz - B - Zellen , die als Vorläufer der reifen B - Lymphozyten 15. Außerdem unter entzündlichen Erkrankungen, medullary Stamm- und Vorläuferzellen wurden in die Milz zu verlagern 16 gezeigt , die Produktion von reifen Monozyten zu induzieren. Dieses Protokoll erlaubt uns nicht, Rückschlüsse auf die Mechanismen zu zeichnen, die bei der Entwicklung von Leukämie und zusätzliche funktionelle sowie molekulare Tests beteiligt sind, so zu tun, eingesetzt werden. Allerdings umfassen diese Daten detaillierte Informationen über die klinischen Merkmale der akuten myelomonocytic Leukämie und hilft Forschern dabei, die Auswirkungen neuer Therapeutika zu bewerten und zu verstehen.

Akute myeloische Leukämie (AML) durch die unkontrollierte Proliferation von hämatopoetischen myeloischen Zellen charakterisiert, die in verschiedenen Stadien der Reifung blockiert sind. Diese Dysregulation kann Einfluss auf die granulocytic, monozytären, erythrozytische oder megaryocytic Differenzierungswege 1. AML-Zellen reichern sich im Knochenmark, zu einer Beeinträchtigung der Hämatopoese führt, die in Thrombopenie, Lymphopenie und Anämie führt. Die leukämischen Zellen dringen auch das Blut und nicht-lymphatischen Organen. Die C1498 - Maus - Modell wird seit Jahrzehnten als Modell für akute Leukämie , da Krebszellen wurden isoliert aus einer leukämischen 10 Monate alte C57BL / 6 (H-2 b) weibliche Maus in 1941. In der Literatur wird die Invasion in das Blut verwendet hämatopoetische Organe (zB die Milz und Lymphknoten) und nicht-hämatopoetischen Organen (beispielsweise der Leber, Lunge, Ovarien und Nieren) durch stark proliferative C1498 - Zellen , nachdem sie wurden über eine in injizierteintravenöse, subkutane oder intraperitoneale Route in empfängliche Mäuse 2-4. Allerdings wurde diese Maus - Modell berichtet entweder granulocytic 2,5 oder myelomonocytic 6 Leukämie zu induzieren. In jüngerer Zeit 7 beschrieben , diese Art von Krebs als murine NKT - Zell - Leukämie eine Studie in 2002 veröffentlicht. Somit unterscheidet sich die Literatur die Natur dieser Zellinie C1498 betreffend und die zugehörige Leukämie in Mäusen induziert. Diese Diskrepanzen sind vor allem auf einen Mangel an detaillierten und aktualisierten veröffentlichten Informationen über die Zellen und die leukämische Erkrankung im Allgemeinen, weil viele Studien in den 1950er Jahren durchgeführt wurden - 70er Jahre. Hier stellen wir ein detailliertes Protokoll zu beschreiben, wie C1498-Zellen zu charakterisieren und die Art der leukämischen Krankheit zu analysieren, die durch die intravenöse Injektion in Mäuse induziert wird. Der erste Abschnitt dieses Protokolls wird auf eine Beschreibung der C1498 - Zellen gewidmet , die in vitro kultiviert wurden. Fluorescent Antiunter Verwendung von Durchflusszytometrie gerichtet Körper gegen die Oberfläche und die intrazelluläre wurden hämatopoetischen Marker verwendet ihren Phänotyp zu bestimmen. Die Anwesenheit von Myeloperoxidase beurteilt wurde Immunfluoreszenz-Mikroskopie unter Verwendung ihrer hämatopoetischen Abstammungslinie und Differenzierungsstadium wurden mit Zytochemie ausgewertet, die Aktivität von Esterasen zu bewerten und May-Grünwald-Giemsa-Färbung wurde durchgeführt. Die C1498-Zellen wurden dann in Mäuse injiziert und die akute Leukämie Krankheit, die induziert wurde, ist in dem zweiten Abschnitt des Manuskripts beschrieben. Die gleichen Techniken wurden verwendet, um die Frequenzen zu bestimmen und die Phänotypen von leukämischen und inhärenten Zellen im Knochenmark, peripherem Blut, Milz und nicht-hämatopoetischen Organen (Leber und Lunge). Dieses Protokoll ist in hohem Maße reproduzierbar, und die hier vorgestellten Daten werden Forschern helfen, die Auswirkungen von neuen therapeutischen Strategien zu bewerten. Diese Leukämie-Maus-Modell wurde bereits für die Immuntherapie zu testen, nähert sich einnd verschiedenen Krebschemotherapeutika 8,9. Ihre Wirksamkeit wurde durch die Bestimmung der Entwicklung der Tumorlast und Überlebensraten beurteilt. Dieses Protokoll kann verwendet werden, zusätzliche Informationen über die Verteilung zur Verfügung zu stellen und Aufenthaltskosten von leukämischen und anderen hämatopoetischen Zellpopulationen während der Behandlung.

<table><tbody><tr><td>C1498 cell line</td><td>ATCC</td><td>TIB 49</td><td></td></tr><tr><td>C57BL/6J-Ly5.1</td><td>Charles River</td><td>B6.SJL-Ptprc a Pep3 b/BoyCrl</td><td></td></tr><tr><td>&lt;strong&gt;Cells culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Mercaptoethanol, Gibco</td><td>ThermoFisher Scientific</td><td>21985</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher Scientific</td><td>10270</td><td></td></tr><tr><td>HEPES, Gibco (1 M)</td><td>ThermoFisher Scientific</td><td>15630</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution, Gibco&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11140</td><td></td></tr><tr><td>Penicillin-Streptomycin, Gibco&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15140</td><td></td></tr><tr><td>RPMI 1640 Medium (Gibco, GlutaMAX Supplement)</td><td>ThermoFisher Scientific</td><td>61870</td><td></td></tr><tr><td>Sodium Pyruvate, Gibco (100 mM)</td><td>ThermoFisher Scientific</td><td>11360</td><td></td></tr><tr><td>&lt;strong&gt;Flow cytometry staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse B220 APC (1)</td><td>ebiosciences</td><td>17-0452</td><td>clone RA3-6B2, final dilution 1/100</td></tr><tr><td>anti-mouse B220 biotin (2)</td><td>ebiosciences</td><td>13-0452</td><td>clone RA3-6B2, 1/400</td></tr><tr><td>anti-mouse CD3 eFluor450 (1)</td><td>ebiosciences</td><td>48-0032</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE (2)</td><td>BD biosciences</td><td>555275</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE-Cy5 (3)</td><td>ebiosciences</td><td>15-0031</td><td>clone 145-2C11, 1/100</td></tr><tr><td>anti-mouse CD4 APC (1)</td><td>ebiosciences</td><td>17-0041</td><td>clone GK1.5, 1/500</td></tr><tr><td>anti-mouse CD4 PE (2)</td><td>ebiosciences</td><td>12-0041</td><td>clone GK1.5, 1/200</td></tr><tr><td>anti-mouse CD8 biotin (1)</td><td>ebiosciences</td><td>13-0081</td><td>clone 53-6.7, 1/100</td></tr><tr><td>anti-mouse CD8 eFluor450 (2)</td><td>ebiosciences</td><td>48-0081</td><td>clone 53-6.7, 1/500</td></tr><tr><td>anti-mouse CD11a biotin</td><td>ebiosciences</td><td>13-0111</td><td>clone M17/4, 1/100</td></tr><tr><td>anti-mouse CD11b PE&amp;nbsp;</td><td>ebiosciences</td><td>12-0112</td><td>clone M1/70, 1/200</td></tr><tr><td>anti-mouse CD16/32 biotin</td><td>ebiosciences</td><td>13-0161</td><td>clone 93, 1/400</td></tr><tr><td>purified anti-mouse CD16/32 (FcR blocking)&amp;nbsp;</td><td>BD biosciences</td><td>553141</td><td>clone 2.4G2</td></tr><tr><td>anti-mouse CD18 biotin (1)</td><td>ebiosciences</td><td>13-0181</td><td>clone M18/2, 1/100</td></tr><tr><td>anti-mouse CD18 FITC (2)</td><td>ebiosciences</td><td>11-0181</td><td>clone M18/2, 1/50</td></tr><tr><td>anti-mouse CD19 PE</td><td>ebiosciences</td><td>12-0193</td><td>clone 1D3, 1/200</td></tr><tr><td>anti-mouse CD21/35 PE</td><td>ebiosciences</td><td>12-0211</td><td>clone 8D9, 1/50</td></tr><tr><td>anti-mouse CD31 PE</td><td>ebiosciences</td><td>12-0311</td><td>clone 390, 1/100</td></tr><tr><td>anti-mouse CD34 eFluor660</td><td>ebiosciences</td><td>50-0341</td><td>clone RAM34, 1/20</td></tr><tr><td>anti-mouse CD44 PE</td><td>BD biosciences</td><td>553134</td><td>clone IM7, 1/50</td></tr><tr><td>anti-mouse CD45.2 FITC&amp;nbsp;</td><td>ebiosciences</td><td>11-0454</td><td>clone 104, 1/100</td></tr><tr><td>anti-mouse CD49b/Pan NK PE</td><td>&amp;nbsp;BD biosciences</td><td>553858</td><td>clone DX5, 1/50</td></tr><tr><td>anti-mouse CD80 biotin</td><td>ebiosciences</td><td>13-0801</td><td>clone 16-10A1, 1/200</td></tr><tr><td>anti-mouse CD86 biotin</td><td>ebiosciences</td><td>13-0862</td><td>clone GL1, 1/200</td></tr><tr><td>anti-mouse CD107b (Mac-3) PE</td><td>ebiosciences</td><td>12-5989</td><td>clone M3/84, 1/40</td></tr><tr><td>anti-mouse CD115 PE</td><td>ebiosciences</td><td>12-1152</td><td>clone AFS98, 1/100</td></tr><tr><td>anti-mouse CD117 eFluor450</td><td>ebiosciences</td><td>48-1171</td><td>clone 2B8, 1/100</td></tr><tr><td>anti-mouse CD127 PE</td><td>ebiosciences</td><td>12-1271</td><td>clone A7R34, 1/100</td></tr><tr><td>anti-mouse CD150 APC</td><td>ebiosciences</td><td>17-1501</td><td>clone 9D1, 1/20</td></tr><tr><td>anti-mouse CD274 (PD-L1) biotin</td><td>ebiosciences</td><td>13-5982</td><td>clone MIH5, 1/200</td></tr><tr><td>anti-mouse Ly-6A/E (Sca-1) PE</td><td>ebiosciences</td><td>12-5981</td><td>clone D7, 1/100</td></tr><tr><td>anti-mouse Ly-6C APC</td><td>ebiosciences</td><td>17-5932</td><td>clone HK1.4, 1/200</td></tr><tr><td>anti-mouse Ly-6G&amp;nbsp; (Gr-1) biotin (1)</td><td>ebiosciences</td><td>13-5931</td><td>clone RB6-8C5, 1/400</td></tr><tr><td>anti-mouse Ly-6G FITC (2)</td><td>ebiosciences</td><td>11-9668</td><td>clone 1A8, 1/50</td></tr><tr><td>anti-mouse MHC class I (H-2Db) biotin</td><td>ebiosciences</td><td>13-5999</td><td>clone 28-14-8, 1/50</td></tr><tr><td>anti-mouse MHC class II (I-A/I-E) PE-Cy5</td><td>ebiosciences</td><td>15-5321</td><td>clone M5/114.15.2, 1/1000</td></tr><tr><td>anti-mouse NK1.1 PE</td><td>BD biosciences</td><td>557391</td><td>clone PK136, 1/50</td></tr><tr><td>anti-mouse TCRVb FITC</td><td>BD biosciences</td><td>553170</td><td>clone H57-597, 1/50</td></tr><tr><td>streptavidin PE-Cy5</td><td>ebiosciences</td><td>15-4317</td><td>&amp;nbsp;&amp;nbsp; 1/200</td></tr><tr><td>Immunofluorescence staining reagents</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse myeloperoxidase (heavy chain antibody)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>sc-16129</td><td>1/10 (20 &amp;micro;g/ml)</td></tr><tr><td>&amp;nbsp;anti-goat IgG (Texas Red coupled antibody)</td><td>Jackson Immunoresearch</td><td>705-076-147</td><td>1/250</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immunoresearch</td><td>017-000-001</td><td></td></tr><tr><td>Hoechst solution</td><td>BD Biosciences</td><td>561908</td><td>1/1000</td></tr><tr><td>Mounting medium 1 (Fluoromount)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4680</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>&lt;strong&gt;Esterase cytochemical staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Naphtol AS-D chloroacetate solution</td><td>Sigma-Aldrich</td><td>911</td><td></td></tr><tr><td>Dye solution (Fast Red Violet LB Base solution)</td><td>Sigma-Aldrich</td><td>912</td><td></td></tr><tr><td>pH6.3 buffer concentrate (TRIZMAL)</td><td>Sigma-Aldrich</td><td>913</td><td></td></tr><tr><td>Sodium nitrite solution</td><td>Sigma-Aldrich</td><td>914</td><td></td></tr><tr><td>Citrate solution</td><td>Sigma-Aldrich</td><td>915</td><td></td></tr><tr><td>Hematoxylin solution</td><td>Sigma-Aldrich</td><td>GHS3</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Formaldehyde solution, 37%</td><td>Sigma-Aldrich</td><td>F1635</td><td></td></tr><tr><td>&amp;alpha;-naphthyl butyrate solution</td><td>Sigma-Aldrich</td><td>1801</td><td></td></tr><tr><td>Phosphate buffer solution</td><td>Sigma-Aldrich</td><td>1805</td><td></td></tr><tr><td>Sodium nitrite Tablet</td><td>Sigma-Aldrich</td><td>1809</td><td></td></tr><tr><td>Pararosaniline solution</td><td>Sigma-Aldrich</td><td>1804</td><td></td></tr><tr><td>Methylene blue solution</td><td>Sigma-Aldrich</td><td>1808</td><td>1/10</td></tr><tr><td>mounting medium 2 (Clearmount)</td><td>Invitrogen</td><td>00-8010</td><td></td></tr><tr><td>&lt;strong&gt;May-Gr&amp;uuml;nwald Giemsa staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>May-Gr&amp;uuml;nwald solution</td><td>RAL Diagnostics</td><td>320070</td><td></td></tr><tr><td>Giemsa R solution&amp;nbsp;</td><td>RAL Diagnostics</td><td>320310</td><td>&amp;nbsp;&amp;nbsp; 1/20</td></tr><tr><td>pH 6.8 buffer solution</td><td>RAL Diagnostics</td><td>330368</td><td></td></tr><tr><td>&lt;strong&gt;Others materials, reagents and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketamine 1000 (100 mg/ml)</td><td>VIRBAC</td><td>3597132111010</td><td></td></tr><tr><td>Xylazine SEDAXYLAN (20 mg/ml)</td><td>CEVA</td><td></td><td></td></tr><tr><td>Bovin albumin serum (BSA) powder</td><td>ThermoFisher Scientific</td><td>BP671</td><td></td></tr><tr><td>PBS solution (1x concentrate)</td><td>ThermoFisher Scientific</td><td>14190</td><td></td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Ultra Pure 0.5 M EDTA solution, pH 8.0</td><td>ThermoFisher Scientific</td><td>15575</td><td></td></tr><tr><td>Separating solution (Pancoll Mouse)</td><td>PANBIOTECH</td><td>P04-64100</td><td></td></tr><tr><td>Lysis buffer (Red Blood Cells Lysing Buffer) (10x)</td><td>BD Biosciences</td><td>555899</td><td>1/10</td></tr><tr><td>NaOH 10 N</td><td>ThermoFisher Scientific</td><td>SS267</td><td></td></tr><tr><td>Trypan blue solution (0.4 %)</td><td>ThermoFisher Scientific</td><td>15250-061</td><td>1/2</td></tr><tr><td>50 ml tube (Falcon)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer (Falcon)&amp;nbsp;</td><td>Corning Life Sciences</td><td>352350</td><td></td></tr><tr><td>&amp;nbsp;Chamber &amp;amp; filter card (EZ Cytofunnel Shandon)</td><td>Thermo Scientific</td><td>A78710003</td><td></td></tr><tr><td>Microscope Cover Glasses, 24 x 24mm</td><td>Knittel Glass</td><td>VD1 2424 Y100</td><td></td></tr><tr><td>Slides (Starfrost - ground edges 90)</td><td>Knittel Glass</td><td>VS1137# 077FKB</td><td></td></tr><tr><td>EDTA Tube</td><td>Greiner Bio-One</td><td>454034</td><td></td></tr><tr><td>Pasteur Pipette 150 mm (capillary tube)</td><td>Fisherbrand</td><td>1154-6963</td><td></td></tr><tr><td>26 G needle</td><td>Terumo</td><td>NN-2613R</td><td></td></tr><tr><td>&amp;nbsp;insulin syringe and needle 29 G</td><td>Terumo</td><td>BS05M2913</td><td></td></tr><tr><td>30 G needle&amp;nbsp;</td><td>Becton &amp;amp; Dickinson</td><td>304000</td><td></td></tr><tr><td>Flow cytometry tubes (blue)</td><td>Beckman Coulter</td><td>2523749</td><td></td></tr><tr><td>Water-repellent pen (Dakopen)</td><td>Dako</td><td>S200230</td><td></td></tr><tr><td>Sharp sterile scissors&amp;nbsp;</td><td>Nessi-care</td><td>SCI-01</td><td></td></tr><tr><td>Sterile forceps</td><td>Dominique Dutscher</td><td>956506</td><td></td></tr><tr><td>Thoma cell counting chamber</td><td>VWR</td><td>631-0397</td><td></td></tr><tr><td>Petri Dishes (Fisherbrand Plastic)</td><td>ThermoFisher Scientific</td><td>S33580A</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 ml)</td><td>ThermoFisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Cylindrical Restrainer 15 - 30 gm</td><td>Stoelting</td><td>51338</td><td></td></tr><tr><td>Shandon Cytospin 3 Cytocentrifuge</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SS-10L</td><td></td></tr><tr><td>1 ml syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.08543</td><td></td></tr><tr><td>Sterile gauze sponges</td><td>URGO</td><td>501580</td><td></td></tr></tbody></table>

a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model

Die intravenöse Injektion von C1498-Zellen in syngene oder kongenen Mäusen durchgeführt worden ist seit 1941. Diese Injektionen in der Entwicklung von akuter Leukämie führen. Allerdings hat die Natur dieser Krankheit nicht gut sind in der Literatur dokumentiert. Hier stellen wir ein technisches Protokoll für die Charakterisierung von C1498 - Zellen in vitro und für die Art der induzierten Leukämie in vivo zu bestimmen. Der erste Teil dieses Verfahrens ist, fokussiert die hämatopoetischen Abstammungslinie und dem Stadium der Differenzierung von kultivierten C1498-Zellen auf der Bestimmung. Um dies zu erreichen, wird multi-parametrischer durchflusszytometrische Färbung verwendet, um hämatopoetische Zellmarker erkennen. Immunofluoreszenz-Mikroskopie, Zytochemie und einer May-Grünwald-Giemsa-Färbung werden dann durchgeführt, um die Expression von Myeloperoxidase zu beurteilen, die Aktivität von Esterasen und zellulärer Morphologie, respectively. Der zweite Teil dieses Protokolls wird auf die Beschreibung der Leukämieerkrankung gewidmet , die in induziert wirdvivo. Letzteres kann durch die Bestimmung der Frequenzen von leukämischen und inhärenten Zellen im Blut, hämatopoetischen Organe (zB Knochenmark und Milz) und nicht-lymphoiden Geweben (zB Leber und Lunge) unter Verwendung spezifischer Färbung und Durchflusszytometrie - Analysen erreicht werden. Die Art der Leukämie bestätigt dann in dem Knochenmark für spezifische Esterasen May-Grünwald-Giemsa-Färbung und Färbung verwendet wird. Hier präsentieren wir die Ergebnisse, die erhalten wurden unter Verwendung dieses Protokolls in altersangepassten C1498- und PBS-injizierten Mäusen.

Um die C1498-Mausmodell charakterisieren, gingen wir mit zwei großen Schritten. Zuerst wurden die C1498 - Zellen charakterisiert ihre hämatopoetischen Abstammungslinie und Reifungsstufe in vitro (1) zu bestimmen. Diese Zellen wurden dann in kongenen Mäusen injiziert, und die Art der leukämischen Krankheit induziert wurde beurteilt unterschiedliche Merkmale bestimmen: leukämischen Zellinfiltration, ihren Phänotyp, eine Quantifizierung der hämatopoetischen Zellen (mature und Progenitoren / Precursoren) in Knochenmark, die Frequenzen von C1498-Zellen und reifen hämatopoetischen Zellen im Blut und einer Beurteilung der Organschwellung (in der Milz, Leber und Lunge) und zelluläre Zusammensetzung. Um die C1498 Zellphänotypen in vitro zu charakterisieren, wurden die Zellen mit Antikörpern gegen Molekülen markiert, die von hämatopoetischen Vorläufern und reifen Zellen (Tabelle 1) ausgedrückt werden, und die Ergebnisse wurden unter Verwendung von Strömungs cytomet analysiertry. Die C1498-Zellen waren positiv für Zelloberflächenexpression von Mac-1 (CD11b / CD18) (~ 7%), B220 (&gt; 25%), und sie zeigten eine intrazelluläre Expression von CD3 &amp; egr;, T-Zell-Rezeptor (TCR) Vß Ketten und Mac -3 (2A und B). Die Zellen waren negativ für die Zelloberflächenmarker Ly6G, Ly6C, CD115, CD21 / CD35, CD19, CD3, CD4, CD8, NK1.1 und pan-NK-Moleküle und für die intrazelluläre Expression von CD4 und CD8 (Daten nicht gezeigt) . Sie wurden dann für Marker von hämatopoetischen Stammzellen und Progenitoren (Tabelle 1) untersucht. Sie waren auch negativ für Zelloberflächenexpression von CD117, CD34, Sca-1, CD150 und CD16 / 32 (Daten nicht gezeigt). Diese leukämischen Zellen wurden dann getestet, um die Expression von Adhäsionsmolekülen, die Antigenpräsentation und co-stimulatorische Moleküle zu bestimmen. Die Zellen exprimierten die Oberflächenmarker LFA-1 (CD11a / CD18), CD44, CD31 (PECAM-1), H-2D b und waren negativ für MHC - Klasse II, CD80, CD86 und CD274 (Daten nicht gezeigt). C1498-Zellen therefore ausgedrückt beide myeloischen (Mac-1, Mac-3) und lymphatischen Markern (B220, CD3, TCR). Um besser auf ihre hämatopoetischen Abstammungslinie zu charakterisieren, wurde Myeloperoxidase Expression unter Verwendung von Immunfluoreszenz-Mikroskopie untersucht. Alle Zellen waren positiv für die Myeloperoxidase, die ihre myeloiden Ursprungs (3A) verifiziert. Eine Mehrheit der Zellen auch positiv gefärbt für α-Naphthyl - Butyrat Esterasen (3B, linke Tafel), und einige von ihnen für die Naphthol - AS-D chloroacetate Esterasen (schwarze Pfeile) (3B, rechts) gefärbt. Die Ergebnisse zeigen, dass die Zellen, Mischungen von monozytären und granulozytäre Zellen enthalten. Nach dem May-Grünwald Giemsa durchgeführt wurde, wurden die C1498 - Zellen beobachtet eine Explosion förmige Morphologie mit einem hohen Kern-Zellplasma - Verhältnis angezeigt werden , 3 bis 5 Kernkörperchen im Kern, ein perinukleären Halo, zahlreiche Vakuolen und ein basophilen Zytoplasma (3C ). thuns wird die C1498-Zelllinie von Monoblasten und Myeloblasten zusammen. Die C1498 - Zellen (CD45.2 +) wurden dann intravenös in CD45.1 + injizierten Mäusen. Die Mäuse erlag 17 bis 19 Tage, nachdem die Zellen injiziert wurden. Diese Mäuse wurden getötet, so dass ihre Leukämietyp analysiert werden, bevor sie an der Krankheit gestorben. Die Kontrollmäuse, die mit PBS injiziert wurden, wurden in den gleichen Zeitpunkten zum Vergleich analysiert. Der C1498-Zellen injizierten Mäuse zeigten massive Infiltration von C1498 - Zellen in ihrem Knochenmark, wie es durch den Hochartiges Aussehen der Zellen zeigten nach der May-Grünwald - Giemsa - Färbung (4A) durchgeführt wurde. Sie bewahrt ihre monozytären und granulocytic Phänotypen (4B und C), eine Ansammlung von monoblastischen und myeloische Zellen zeigen , die charakteristisch für akute myelomonozytische Leukämie ist. Um determine ob Markzahlen hämatopoetischen Zellen nach leukämischen Zellen Invasion niedriger waren, CD45.2 + C1498 - Zellen, B lymphatischer, monozytären und granulocytic Populationen (einschließlich Vorläufern, Vorläufern und reifen Zellen) wurden unter Verwendung von Immunfluoreszenzanfärbung quantifiziert und multiparametrisches Durchflusscytometrieanalyse. Leukämischen Zellen 16 bis 36% der hämatopoetischen Zellen dargestellt (Daten nicht gezeigt). Die anderen Zelltypen wurden alle in deutlich niedrigeren Zahlen in den C1498-injizierten Mäusen als in den PBS-injizierten Mäusen (von 5-fach im Durchschnitt für B-Zell-Subpopulationen, 4-fach im Durchschnitt granulocytic Zellen und 3-fach im Durchschnitt für monozytären Subsets) (5A bis C). Eine Untersuchung der Frequenzen von mononukleären Zellen in leukämischen und Kontroll - Maus Blutproben zeigten , daß sie einen vergleichbaren Prozentsatz der Lymphozyten (Figur 6A) , aber eine höhere Frequenz monozytären enthaltenenund leukämischen Zellen. Diese Eigenschaften sind repräsentativ für akute myelomonozytische Leukämie 11 (6B). Zu den weiteren Merkmalen der akuten Leukämie myelomonocytic 12, die C1498-injizierten Mäusen mit geschwollenen Leber (Hepatomegalie) präsentiert, Lunge und Milz (Splenomegalie) (7A). Verschiedene Frequenzen von CD45.2 + C1498 - Zellen wurden in diesen Organen mit Immunfluoreszenzanfärbung erkannt und Durchflusszytometrie - Analyse (7B). Wie Splenomegalie aus hohen Zahl von infiltriert Monozyten führen kann, schätzten wir auch die Anteile von Milz- Populationen. Die Zahl der Zellen in der B - lymphozytische, monozytären und granulocytic Zellfraktionen waren signifikant größer ist , um durchschnittlich 2-fach, 2,5-fach und 3-fach bzw. in leukämischen Milzen als bei den Kontroll Milzen (7C). Thin-page = &quot;1&quot;&gt; <img alt="Abbildung 1" src="/files/ftp_upload/54270/54270fig1.jpg" /> Abbildung 1. Schematische Darstellung des Protokolls Set Up für in - vitro - Charakterisierung von Cultured C1498 Zelllinien und in - vivo - Beschreibungen von akuter Leukämie. Hämatopoetischen Abstammungslinie und dem Differenzierungsstadium von Gewebekultur C1498 - Zellen wurden zuerst bestimmt. C1498-Zellen wurden dann in kongenen Mäusen injiziert, um die Entwicklung von akuter Leukämie zu induzieren. Die Isolierung von Knochenmark, peripherem Blut, Milz, Leber und Lungengewebe wurde durchgeführt, um die Frequenzen, Phänotypen und morphologische Veränderungen nach der C1498-Zellen-Infiltration zu bestimmen. IV: Intravenous MGG. May-Grünwald Giemsa <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <html> ad / 54270 / 54270fig2.jpg &quot;/&gt; Abbildung 2 Phänotypische Analyse von C1498 - Zellen nach in vitro - Kultur. Representative Durchflusszytometrie Punktplots und Histogramme der Zelloberfläche (A) und intrazelluläre (B) C1498-exprimierten Moleküle , die mit hämatopoetischen mature Zelldifferenzierung zugeordnet wurden , sind gezeigt. C1498-Zellen wurden aus den Kulturen geerntet, gewaschen und markiert fluoreszierenden Antikörpern, die für die Zelloberflächen CD11b, CD18 und B220 Marker oder deren Isotypkontrollen spezifisch waren. Für die intrazelluläre Färbung wurden die Zellen fixiert, permeabilisiert und markierten Antikörpern, gerichtet gegen Mac-3, CD3 &amp; egr; und ein gemeinsames Epitop des TCR (T-Cell Receptor) Vß-Kette oder deren Isotyp-Kontrollen verwendet. Die Analysen wurden unter Verwendung von Gating mit lebenden Zellen durchgeführt werden . <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <html> ntent &quot;fo: keep-together.within-page =&quot; 1 &quot;&gt; <img alt="Figur 3" src="/files/ftp_upload/54270/54270fig3.jpg" /> Abbildung 3. Funktionelle und morphologische Charakterisierung von Cultured C1498 Zellen. C1498 - Zellen aus Kulturen und zentrifugiert auf Objektträger für die Mikroskopie. (A) Die Färbung für Myeloperoxidaseexpression geerntet wurden , wurde unter Verwendung von Immunfluoreszenz durchgeführt. (B) Zytochemische Reaktionen wurden verwendet , um die α-naphthyl Butyrat - Esterase (NBE) und Naphthol AS-D chloroacetate Esterase zu analysieren (CAE) Aktivitäten in C1498-Zellen. Die Zellen wurden für jedes Etikett als positiv betrachtet , wenn braun und rot-lila, große zytoplasmatischen Granula bzw. beobachtet. (C) May-Grünwald Giemsa (MGG) Färbung von C1498 - Zellen. Für jede Färbung Experiment wird die Mikroskopie Objektiwergrößerung angezeigt. Jedes Bild ist repräsentativ für drei getrennte Experimente.large.jpg &quot;target =&quot; _ blank &quot;&gt; Bitte hier klicken, um eine größere Version dieser Figur zu sehen. <img alt="Abbildung 4" src="/files/ftp_upload/54270/54270fig4.jpg" /> Abbildung 4. Knochenmark - Morphologien in PBS- und C1498-injizierten Mäusen. Die Knochenmarkzellen von PBS- und C1498 Zellen injizierten Mäusen und zentrifugiert auf Objektträger für die Mikroskopie isoliert. (A) May-Grünwald Giemsa (MGG) Färbung. (B ) α-naphthyl Butyrat - Esterase (NBE) und (C) Naphthol AS-D - Chloracetat - Esterase (CAE) Funktionen wurden mit Zytochemie ausgewertet. Im Feld A, sind die Band (unreife) oder segmentiert (reifen) Neutrophilen weniger sichtbar im Knochenmark der C1498-injizierten Mäusen als die PBS-injizierten Mäusen. Panel B und C zeigen an, daß eine Ansammlung von monocytischen und granulozytäre Zellen in der leukämischen Knochenmark im Vergleich zu den Zahlen in der Kontrollknochenmark beobachtet war. Allemikroskopische Analysen wurden unter Verwendung eines 100 - facher Vergrößerung Ziel durchgeführt. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Abbildung 5" src="/files/ftp_upload/54270/54270fig5.jpg" /> Abbildung 5. Quantitative Analyse von Medulläre Populations in PBS- und C1498-injizierten Mäusen. Die Knochenmarkzellen von PBS- und C1498 Zellen injizierten Mäusen isoliert wurden und geschätzt nach Zellzählung durchgeführt wurde. Die Frequenzen der verschiedenen Zellpopulationen wurden nach Immunfärbung und lebenden Zellen Durchflußcytometrie - Analyse gated bestimmt. (A) Die B - Zell - Untergruppen enthalten CD19 + B220 + -Zellen in Stufen von pro-B - Zellen , B - Lymphozyten (B) granulozytäre Zellen in dem reifen CD3 - und CD11b + Ly6G + Abstammungslinien, die Vorläufer enthalten ein. nd unreifen und reifen Granulozyten (C) Die Monozyten - Subsets wurden als CD3 definiert - CD115 + und enthalten Zellen in der Vorläufer zu Monozyten Stufen reifen. n = 7 Mäuse / Gruppe, und die Daten werden als Histogramme dargestellt, die Mittel ± SEM zeigt. ***, P &lt;0,0001 und ** p &lt;0,01, t - Test verglichen PBS- und C1498-injizierten Mäusen ungepaarten Studenten. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">Bitte klicken Sie hier</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="Figur 6" src="/files/ftp_upload/54270/54270fig6.jpg" /> Abbildung 6. Blut - Analyse von mononukleären Zell - Populationen in PBS- und C1498-injizierten Mäusen. Repräsentative Durchflusscytometrie Punktplots von (A) T und B - Lymphozyten - Prozentsätze, die jeweils definiert wurden als CD3 + und B220 + -Zellen in PBS- und C1498-Zellen injiziert. Mäuse (B) monozytären Zellfrequenzen in C1498 leukämischen und Kontrolle (PBS) Mäuse wurden durch die Analyse von CD115 + Ly6C bestimmt - und CD115 + Ly6C hohe Zellen. Die Analyse wurde durch Gating lebenden Zellen durchgeführt. Zum Vergleich leukämischen und Kontrollmäuse, + CD45.2 C1498 Zellen wurden ausgeschlossen. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <img alt="7" src="/files/ftp_upload/54270/54270fig7.jpg" /> Abbildung 7. Schätzung der Splenic Populations in Leukämische und Kontrollmäusen. (A) Repräsentative Photographien von Leber, Lunge und Milz in leukämischen Mäusen Quellung im Vergleich zu Kontrollmäusen. Die Milzen wurden gesammelt und gewogen, und Splenozyten wurden folgende Gewebezerstörung gezählt. (B) Histogramm darstellt leukämische Zelle Frequenzen in diffErent Organe nach Immunfärbung wurde für CD45.2 + Zellen durchgeführt , und die Ergebnisse wurden mittels Durchflusszytometrie analysiert fließen. (C) Ermittlung der Milz - B, granulozytärer und monozytären Zellzahlen nach Immunfärbung und Durchflusszytometrie - Analyse Gating durchgeführt wurden live CD19 + B220 zu identifizieren + , CD3 - CD11b + Ly6G +, CD3 - CD11b + Ly6C - und CD3 - CD11b + Ly6C hohe Zellen. Die Maßstabsbalken für die Lunge, Milz und Leber zeigen an, 1 cm. .. n = 5 bis 8 Mäuse / Gruppe, und die Daten werden in Histogrammen als Mittelwert ± SEM dargestellt *, p &lt;0,05; **, p = 0,0033, ungepaarten Student-t- Test Vergleich PBS- und C1498-injizierten Mäusen <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">Bitte hier klicken</a> , um <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">eine größere Version dieser Figur zu sehen.</a> <table fo:keep-together.within-page="1"><tbody&gt; <tr><td> Zelltyp </td><td> Membran oder intrazelluläre Moleküle </td></tr><tr><td></td><td></td></tr><tr><td> Precursors und reifen Zellen </td><td></td></tr><tr><td> NK-Zellen </td><td> NK1.1 +, pan-NK + </td></tr><tr><td> NKT-Zellen </td><td> NK1.1 +, pan-NK +, TCR Vbeta + (8.2), CD3 + </td></tr><tr><td> T-Lymphozyten </td><td> TCR Vbeta +, CD3 +, CD4 +, CD8 + </td></tr><tr><td> B-Zellen-Vorläufer und B-Lymphozyten </td><td> B220 +, CD19 +, CD21 / 35 + </td></tr><tr><td> granulocytic Vorläufer und Granulozyten </td><td> Ly6G +, Mac-1 +, CD11b + </td></tr><tr><td> Monozyten-Vorläufern und Monozyten / Makrophagen </td><td> CD11b +, Mac-1 +, Mac-3 +, CD21 / 35 +, CD115 +, Ly6Chi </td></tr><tr><td></td><td></td></tr><tr><td> Stammeltern </td><td></td></tr><tr><td> multi- Vorläufern </td><td> CD117 + Sca-1 + CD34 + (Lin- CD150-) </td></tr><tr><td> lymphatischen grundierten multi- Vorläufern </td><td> CD117hi Sca-1hi CD127 + (Lin-) </td></tr><tr><td> gemeinsame lymphatischen Vorläufern </td><td> CD117lo Sca-1LO CD127 + (Lin-) </td></tr><tr><td> gemeinsame myeloischen Vorläufern </td><td> CD16 / 32lo CD117 + CD34int (Lin- Sca-1-) </td></tr><tr><td> Granulozyten-Makrophagen-Vorläufern </td><td> CD16 / 32hi CD117 + CD34hi (Lin- Sca-1-) </td></tr><tr><td> Megakaryozyten-erythroiden Vorläufern </td><td> CD16 / 32lo CD117 + CD34lo (Lin- Sca-1-) </td></tr><tr><td></td><td></td></tr><tr><td> Hämatopoetischen Stammzellen </td><td> CD117 + Sca-1 + CD150 + (Lin- CD34) </td></tr></tbody></table> Tabelle 1 Marker hämatopoetischer Zelllinien und Differenzierung. CD: Cluster der Differenzierung; Lin: Marker von reifen Zellen; lo:niedrige Expression; hallo: hohe Expression; int: Zwischen Ausdruck; NK: natürliche Killerzellen; TCR: T-Zell-Rezeptor.

Watch this Scientific Journal Video about A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model at JoVE.com

A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Diese Handschrift stellt ein technisches Verfahren , das verwendet werden kann , C1498 - Zellkulturen in vitro und der akuten Leukämie induziert in Mäusen nach der Injektion zu charakterisieren. Die phänotypische und funktionelle Analysen werden mit Hilfe der Durchflusszytometrie, Immunfluoreszenzmikroskopie, Zytochemie und May-Grünwald Giemsa Färbung durchgeführt.

Ein detailliertes Protokoll der murinen C1498-Zelllinie und der damit verbundenen Leukämie Maus-Modell für Charakterisieren

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

a-detailed-protocol-for-characterizing-murine-c1498-cell-line-its

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

19.9K Aufrufe.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC).  Diese Handschrift stellt ein technisches Verfahren , das verwendet werden kann , C1498 - Zellkulturen in vitro und der akuten Leukämie induziert in Mäusen nach der Injektion zu charakterisieren. Die phänotypische und funktionelle Analysen werden mit Hilfe der Durchflusszytometrie, Immunfluoreszenzmikroskopie, Zytochemie und May-Grünwald Giemsa Färbung durchgeführt. 

Serie: A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Balmer Series

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit around the nucleus. Importantly, an atom cannot discharge energy while its electrons are in stationary states. An electron can only emit energy by changing energy states. To change energy states, an electron must move from one orbit to another by either absorbing or emitting energy. This change can only occur if the absorbed or emitted energy equals the difference between the two states. Electrons cannot exist between orbits.
The quantum number, n, is used to label the different energy states. The lowest energy state is the ground state, which is n equal to one. The excited states are labeled n equal to 2, 3, 4, and so on. When an electron at the ground state absorbs a photon whose energy equals the difference between the ground and second state, the electron becomes excited and transitions from the ground state to the n= 2 excited state. If the energy of the photon equals the difference between the ground and third state, the electron moves to the n=3 state.
According to BohrÃÂ¢ÃÂÃÂs model, the potential energy of an electron in the nth level can be calculated using the following equation:
<img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/54/54_Concepts_1.jpg" alt="Hydrogen atom energy levels, En=-Rhc/n² equation, spectroscopy concept, educational diagram." data-altupdatedat="1747830616000" data-alt="Bohr model energy level equation \(E_n = -\frac{Rhc}{n^2}\), quantum mechanics concept.">
where En is the potential energy, R is the Rydberg constant (1.0974 ÃÂÃÂ 107 m-1), h is Planck's constant (6.62607004 ÃÂÃÂ 10-34 m2ÃÂÃÂ·kg/s), and c is the speed of light (~ 3 ÃÂÃÂ 108 m/s). Electrons can also spontaneously return to the ground state or any other lower excited state. When this happens, the excess energy is released in the form of an emitted photon. The energy of the photon is equal to the energy difference between the higher and lower energy states. That energy corresponds to wavelengths of light. Since each type of atom has different energy levels, the light emitted from each transition varies for each atom. For a sample of mixed molecules, the emitted light contains a range of wavelengths in what is called a continuous spectrum. For a sample containing atoms of a single element, the emitted light contains only certain wavelengths, which can be viewed as discrete lines once separated by a prism.
<h4>The Hydrogen Atom</h4>
Looking specifically at the hydrogen atom, the excitation of its electrons requires the absorption of sufficient energy to split the bond in the diatomic molecule H2. Since more energy is used to split the molecule than needed, the electrons in the hydrogen atom absorb the excess energy and are excited to a higher energy level. When the electrons spontaneously return to a lower energy level, light is emitted, which corresponds to the energy difference between the excited level and lower level.
When discussing the emission of energy, the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. The wavelengths of emitted light ultimately depend on the energy difference between the two levels.
In a pure sample of hydrogen gas, the emission spectrum appears as distinct lines of discrete wavelengths that are specific to the element hydrogen. Some of these lines are in the visible range of the electromagnetic spectrum, while some are in the ultraviolet or infrared range.
<h4>The Balmer Series</h4>
The series of visible lines in the hydrogen atom spectrum are named the Balmer series. This series of spectral emission lines occur when the electron transitions from a high-energy level to the lower energy level of n=2. Johann Balmer observed these spectral lines at 410.2 nm, 434.1 nm, 486.1 nm, and 656.3 nm, which correspond to transitions from the n=6, n=5, n=4, and n=3 energy levels to the n=2 level, respectively.
Balmer was able to relate these wavelengths of emitted light using the Balmer formula.
<img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/54/54_Concepts_2.jpg" alt="Wavelength equation: λ=C(m²/(m²−n²)), optical physics formula, educational reference." data-altupdatedat="1747830616000" data-alt="Calculating wavelength using interference equation, formula involving Î», mÂ², and nÂ².">
Here, ÃÂÃÂ» is the observed wavelength, C is a constant (364.50682 nm), n is the lower energy level with a value of 2, and m is the higher energy level, which has a value greater than 3. This observation was then refined by Johannes Rydberg, where R is the Rydberg constant.
<img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/54/54_Concepts_3.jpg" alt="Rydberg formula equation, atomic transitions, spectroscopy, wavelength calculation, physics." data-altupdatedat="1747830616000" data-alt="Hydrogen spectral series formula, equation, Balmer series, wavelength calculation, physics concept.">
Remember that this equation describes emitted light, so the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. In the case of the Balmer series, nf is equal to 2. This equation was combined with the Bohr model to calculate the energy needed to move an electron between its initial and final energy levels, ÃÂÃÂE.
<img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/54/54_Concepts_4.jpg" alt="Hydrogen atom energy level transition formula, ΔE=Rhc(1/n²f−1/n²i), depicting quantized energy." data-altupdatedat="1747830616000" data-alt="Energy transition formula, ÎE=Rhc(1/nfÂ²-1/niÂ²), equation for spectral line calculations.">
Later, other spectral series for the hydrogen atom were discovered. For example, the Lyman series contains emission lines with energies in the ultraviolet region.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silderberg, M.S. (2009). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill.</li>
</ol>

The Hydrogen Atom: Bohr Model and Balmer Series - Concept

Balmer Serie

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit ...

Lehre

JoVE Lab Manual

Chemie

Genetics of Organisms

<h4>Mendelian Genetics</h4>
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this process as changes in the frequency of alleles at individual genetic loci. This simple representation of how evolution occurs dates to Gregor Mendel&#8217;s analysis of trait inheritance patterns in pea plants, first presented in 1865. Mendel determined, using rigorous collection of data, that noticeable traits are controlled by two alleles of each gene (he called them factors), one from each parent. In addition, he concluded that some alleles are dominant to others (deemed recessive). Interestingly, Mendel&#8217;s data was not appreciated until 1900, probably in part because his approach to the study of inheritance was so divergent from his predecessors. Even after his brilliant experiments were rediscovered, it took 30 additional years for Mendelian inheritance to become integrated with evolutionary theory and to gain universal acceptance.
Why was such a seemingly clear and straightforward process dormant for so long and then the center of controversy for decades? One key to the controversy is that we rarely see the clear patterns of inheritance described by Mendel. Animal and plant breeders most often see evidence for what was described as blending inheritance, or offspring whose phenotypes are intermediate between those of their parents, rather than the discrete segregation of traits into well-defined categories, like smooth versus wrinkled peas or red versus white flowers. Mendelian inheritance thus did not align well with practical experience and it took decades to reconcile the two. Additionally, there are now known to be modes of inheritance that, at first glance, appear to violate Mendel&#8217;s laws. Deeper investigations into genetics using molecular biology have effectively characterized these novel modes of inheritance. The new information does not invalidate Mendel&#8217;s findings, however, it simply enhances his model.
<h4>Phenomena that Violate Mendelian Rules</h4>
There are three main phenomena: epistasis, pleiotropy, and sex-linkage that appear to violate the basic dominance and recessivity rules of Mendelian inheritance. Epistasis occurs when two or more genes or alleles interact to affect a phenotype. A function of one gene product may be required to allow another gene to be expressed or to function normally. On the other hand, pleiotropy occurs when one gene controls the expression of multiple phenotypes in an individual. For example, a large proportion of cats with white fur and blue eyes are deaf 1. Pleiotropy is often antagonistic, meaning that the same gene causes beneficial changes in one facet of an individual&#8217;s phenotype while at the same time causing damaging changes in some other aspect of an individual&#8217;s phenotype. Antagonistic pleiotropy is seen as a tradeoff or constraint on evolution, as proposed by George C. Williams. For example, the age specific decline in performance, or senescence, is thought to be a pleiotropic trait controlled by the same genes that increase early-life fecundity2,3.
<h4>Sex Linkage</h4>
Sex linkage is another phenomenon discovered as a counter-example to basic Mendelian inheritance. In many organisms, pairs of sex chromosomes determine gender. In humans and many mammals, XX individuals are female while XY individuals are male. Male offspring inherit an X from their mother and a Y from their father; females inherit an X from their mother and an X from their father. In most species, the ratio of males to females at birth under &#8220;normal&#8221; environmental conditions is 1:1. Y-chromosomes undergo little recombination with X chromosomes during meiosis, due to the fact that, over evolutionary time, they have lost a substantial number of genes. Interestingly, there are only 16 genes shared by the human X and Y chromosomes4.
For X-linked traits (traits encoded by genes on the X chromosome) females can be homozygous (having two copies of the same allele) or heterozygous (having two different alleles). If the trait is recessive, females will only show the phenotype for the trait if they are homozygous. Heterozygous females &#8220;carry&#8221; the recessive allele, but do not express the phenotype of that allele. Statistically speaking, half of her sons will inherit this recessive allele and all will express the phenotype because males have only one X chromosome.
A commonly used example of an X-linked phenotype is hemophilia, a disease that prevents the synthesis of blood clotting proteins. If a female is heterozygous (is a carrier, genotype XH Xh) mates with a normal male (genotype XH Y), 50% of the sons will be affected. If the female was instead homozygous for the trait (genotype Xh Xh ), none of her daughters would be hemophiliacs but 100% of her sons would be. The differential phenotypic ratios seen in sons and daughters appears to violate Mendel&#8217;s laws, yet Mendel&#8217;s first law still applies; each offspring has a 50% chance of inheriting an X-chromosome carrying the hemophilia allele from a heterozygous mother5.
Like humans, the fruit fly Drosophila melanogaster has an XY-sex determination system. D. melanogaster make excellent laboratory organisms because they are easy to keep, breed, and manipulate. Wild-type flies have what is considered normal body morphology and red eyes. Many lines are readily available for purchase, including some with mutations resulting in flies without eyes, with eyes of varying color, or missing wings. These organisms can be used to study or demonstrate both Mendelian and non-Mendelian inheritance patterns. In fact, 1933 Nobel Prize winner, Thomas Hunt Morgan, first noticed that some genes are sex-linked using this system6.
<h4>References</h4>
<ol class="references">
	<li>Strain, George M., &#39; Cat Breeds With Congenital Deafness,&#39; [Online]. Available: https://www.lsu.edu/deafness/catbreeds.htm. [Accessed 29 August 2018].</li>
	<li>W. GC., &#39;Pleiotropy, natural selection, and the evolution of senescence.,&#39; Evolution, vol. 11, p. 398&#8211;411, 1957.</li>
	<li>T. &#38; S. P. S. Flatt, &#39;Integrating Evolutionary and Molecular Genetics of Aging,&#39; Biochimica et Biophysica Acta, vol. 1790, no. 10, p. 951&#8211;962, 2009.</li>
	<li>D. Bachtrog, &#39;Y chromosome evolution: emerging insights into processes of Y chromosome degeneration,&#39; Nature Reviews. Genetics, Vols. 14(2), 113&#8211;124, 2013.</li>
	<li>US National Library of Medicine, &#39;Hemophilia,&#39; Genetics Home Reference, [Online]. Available: https://ghr.nlm.nih.gov/condition/hemophilia. [Accessed 31 August 2018].</li>
	<li>&#39;The Nobel Prize in Physiology or Medicine 1933,&#39; [Online]. Available: https://www.nobelprize.org/prizes/medicine/1933/summary/. [Accessed 31 August 2018].</li>
</ol>

Genetics of Organisms: REF and WEF Fly Genetics - Concept

Genetik der Organismen

Mendelian Genetics
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this ...

Biologie

Genetik

Animal Behavior

<h4>Drosophila as a Model Organism</h4>
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly recognized as a fruit-loving pest. There are multiple reasons that make D. melanogaster an excellent experimental organism: their roughly two-week generation time allows studying multiple generations, they are easily maintained in very small tubes, and their sexual dimorphism enables investigators to easily distinguish between males that have black coloration towards the posterior and females that have banded abdomens.
Thomas Hunt Morgan and his students were among the first to make use of Drosophila, in the early 20th century. In 1933, Morgan received a Nobel Prize for his discovery of the role of chromosomes in heredity, which was founded on his observations of mutant Drosophila. Morgan continued his studies by generating new mutants with radiation. The presence of multiple mutants with discrete effects on the appearance of the fly enabled him to look at patterns of multifactor inheritance. A key aspect of his early work was the discovery of apparent violations of Mendel&#8217;s second law, which states that genes segregate independently of one another. Morgan&#8217;s observations indicated that genes will not segregate independently if they are joined together on the same chromosome. Furthermore, it became possible to determine gene locations on a chromosome relative to each other through analyses of how often they recombined thus triggering gene mapping studies to identify the location of a gene and distances between genes. Consequently, associates of Morgan&#8217;s lab received two additional Nobel Prizes for other studies of Drosophila genetics. Morgan&#8217;s success and the accumulating information about Drosophila inspired others to adopt them as experimental organisms, which added to our knowledge of them and progressively increased their value as a model organism. Other scientists have developed many mutations. Over 27,000 unique Drosophila lines are now maintained as stock cultures at the Drosophila stock centers and are readily available to laboratories that use them as tools for studying diverse aspects of biology, including behavior1.
<h4>Drosophila Ethology</h4>
Animal behavior, or ethology, is the study of how animals interact with each other and their environments. Although it is a relatively young biological field, ethology has important implications since an organism&#8217;s survival depends on how it behaves with regard to possible mates, food sources, and natural obstacles. Studying these organisms helps scientists to understand the contexts of these behaviors and the underlying mechanisms as to how they have evolved. Thus, a goal of the study of animal behavior is to find a genetic basis for the behaviors exhibited by individuals or groups of individuals. For example, kin selection, first proposed by William Hamilton, suggests that individuals will behave altruistically when making a sacrifice for a family member rather than a stranger2. This is because related individuals share more genes than unrelated ones, and the future reproduction by their relatives contributes in a way to their own fitness, or reproductive success. Hence, observing how organisms like Drosophila interact with their environment can allow us to understand how these behaviors are hard-wired. Additionally, these behaviors provide insight into how these organisms have adapted to cope with their environment and survive.
Directional behaviors are commonly studied to understand the basis of how and why an organism moves in its environment. Kinesis and taxis are two forms of directional behaviors. Kinesis involves stimulus-induced movements in random directions, such as the random movement of flies when chased away. On the other hand, taxis involves stimulus-induced movement towards a specific direction, such as moths flying towards a lightbulb. Taxis behaviors are positive if the animal moves towards the stimulus and negative if the animal moves away from the stimulus. Furthermore, taxis behaviors are named based on the specific stimulus that induces them. For example, geotaxis is a response to gravity, where positive geotaxis means that the organism moves with gravity. Phototaxis is movement in response to light, such as positive phototaxis of moths towards a lightbulb. Similarly, chemotaxis is attraction to or avoidance of an airborne chemical cue, such as the negative chemotaxis of many predators, including humans, in response to the smell of skunk spray. Furthermore, there are many other types of taxes that are studied by scientists, including aerotaxis, barotaxis, hydrotaxis, and magnetotaxis, that are movements in response to oxygen, pressure, water, and magnetic fields, respectively.
<h4>Study of Drosophila taxis behavior</h4>
A common and simple method to study Drosophila taxis behavior is to use a choice chamber, which allows the observation of a directional response of the fly to a particular stimulus, such as gravity, light, or a chemical. Drosophila are inserted in the center of the choice chamber and then allowed to wander freely about the chamber, which is designed so that alternative stimuli can be presented at opposing sides. After a period of wandering, the number of flies is counted on either side of the choice chamber. The number of flies on each side of the chamber is then analyzed with the Chi Square test to gauge whether or not there is a preference for a stimulus.
Comparison of directional behavior of mutant and wild-type Drosophila allows researchers to determine the role of the mutated gene in that specific behavior. Moreover, scientists are also trying to understand how animals decide what to do in the presence of combinations of stimuli, including conflicting stimuli3. Together, these studies can help predict how organisms will react to certain stimuli or changes in environmental factors.
<h4>References</h4>
<ol class="references">
	<li>Cook KR, Parks AL, Jacobus LM, Kaufman TC, Matthews K. New research resources at the Bloomington Drosophila Stock Center. Fly. 2010, Vol. 4, 1: 88-91.</li>
	<li>WD, Hamilton. Altruism and related phenomena, mainly in social insects. Ann Rev Ecol Systematics. 1972, Vol. 3, 193-202.</li>
	<li>Gepner R, Skanata MM, Bernat NM, Kaplow M, Gershow M. Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration. eLife. 2015, Vol. 4, e06229.</li>
</ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Concept

Tierverhalten

Drosophila as a Model Organism
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly ...

Ökologie

Hardy-Weinberg and Genetic Drift

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are therefore very difficult to measure. In general, studying phenomena like this requires the use of mathematical models which are built using parameters that can be conveniently measured. These models are then used to make predictions about how changes to the system might affect the outcome. 
For example, changes to the frequency of genetic alleles at individual loci in species are often observed over long time periods, but are not generally observable over short time periods. The use of computer models allows researchers to predict changes within a species&#8217; gene pool using observations that have already been gathered, and allow for simulations of a potentially unlimited number of generations of the population. This would certainly not be possible within a human lifetime.
<h4>The Hardy-Weinberg Equilibrium Principle</h4>
One equation used to model populations is the Hardy-Weinberg equilibrium equation. It was formulated independently in 1908 by both G. H. Hardy and Wilhelm Weinberg1,2. The simple equation describes the expected allele frequency of a population that is not evolving. Because most real-life populations are evolving in response to the forces of natural selection, this formula acts as a useful null hypothesis. It is used to test whether or not different types of selection are, or are not, occurring. If measured allele frequencies differ from those that were predicted using the Hardy-Weinberg equation, then the gene in question is undergoing evolutionary change.
The Hardy-Weinberg equation can be represented as p2 + 2pq + q2 = 1 where p represents the frequency of the dominant allele and q represents the frequency of the recessive allele for the gene in question. Each expression in the equation represents the predicted frequency of one of the three possible diploid genotypes. Homozygous dominant frequency is represented by p2, homozygous recessive by q2, and heterozygous by 2pq. If these frequencies are summed, it adds up to 1, which makes sense since the full population had been divided into the three available categories.
It is also possible to describe the population, or gene pool, in terms of the two alleles, without regard to how they are packaged into diploid individuals. This equation is represented as p + q = 1, that is, the frequencies of the dominant and recessive alleles must add up to 1 within the population. Again, this makes sense, since the model includes just two allele possibilities. Notice that the frequency of genotypes in the population is just the quadratic expansion of the frequency of alleles in the population because (p + q)2 = p2 + 2pq + q2.
The Hardy-Weinberg equation, like most other models, requires a series of assumptions. Usefully, if the real-life data differs from the predicted data, it is possible to hypothesize which of the starting assumptions was false. The assumptions of the Hardy-Weinberg equilibrium equations are: 1) the population is very large, 2) the population is closed, meaning that there are no individuals immigrating into or emigrating out of the population, 3) there are no mutations occurring on the gene in question, 4) individuals within the population are mating at random&#8212;individuals are not choosing their mates, 5) natural selection is not occurring. Again, keep in mind that the Hardy-Weinberg equilibrium equation is a null hypothesis. Frequently, real populations violate one or more of these assumptions. The equation is used to determine if and how evolution is happening.
<h4>Genetic Drift</h4>
In a relatively small population, a condition that violates the first Hardy-Weinberg assumption, it is possible for allele frequencies to have resulted from chance. This phenomenon is referred to as genetic drift. One version of this is referred to as the founder effect. If a small number of individuals move to an isolated location and start a new population, the specific genetics of those particular individuals will shape the future generations. This new small gene pool may have the same allele frequency as the original, but it is also possible, even likely, that it does not. Say the original population included 50% dominant alleles and 50% recessive. In the extreme, if all of the individuals in a migrating founder group are homozygous recessive, then the dominant allele has been lost completely and the recessive is now at 100%. A similar phenomenon, called the bottleneck effect, can occur if a population sustains a large drop in numbers due to a natural disaster, human intervention, or disease.
Importantly, genetic drift represents a type of evolution that is not necessarily adaptive. It does not specifically select for traits that are fit for the environment. Nonetheless, it is an important evolutionary force in shaping populations with a small number of individuals and will cause deviations from Hardy-Weinberg predictions. In populations that are larger, deviations from the predictions are more likely to mean that the population in question is undergoing evolution through natural selection. Under these circumstances, further consideration of the mechanisms causing this evolution can be explored.
Modern applications of the Hardy-Weinberg model include analysis of human and animal populations in terms of how their immune systems relate to their susceptibility to infectious disease3,4. Many research groups are cataloging genes which encode specific immune system molecules, such as CCR5 and the major histocompatibility complex (MHC, known in humans as the human leukocyte antigen (HLA), and correlating this information to epidemiological studies regarding disease and its progression3,5-6. From comparing these two types of data sets, patterns are emerging showing that some individuals are genetically resistant to particular infections while others are more likely to succumb to disease4. Hardy and Weinberg&#8217;s model has been essential to the analysis of this data and has contributed to our understanding of how infections have shaped evolution.
<h4>References</h4>
<ol class="references">
	<li>Hardy, G. H. (Jul 1908). 'Mendelian Proportions in a Mixed Population' (PDF). Science. 28 (706): 49&#8211;50. doi:10.1126/science.28.706.49. ISSN 0036-8075. PMID 17779291.</li>
	<li>Weinberg, W. (1908). '&#220;ber den Nachweis der Vererbung beim Menschen'. Jahreshefte des Vereins f&#252;r vaterl&#228;ndische Naturkunde in W&#252;rttemberg. 64: 368&#8211;382.</li>
	<li>Eguchi S, Matsuura M. Testing the Hardy-Weinberg equilibrium in the HLA system. Biometrics. 1990 Jun;46(2):415-26. PubMed PMID: 2364131.</li>
	<li>Phillips KP, Cable J, Mohammed RS, et al. Immunogenetic novelty confers a selective advantage in host-pathogen coevolution. Proc Natl Acad Sci U S A. 2018;115(7):1552-1557.</li>
	<li>Lim JK, Glass WG, McDermott DH, Murphy PM CCR5: no longer a 'good for nothing' gene--chemokine control of West Nile virus infection. Trends Immunol. 2006 Jul; 27(7):308-12.</li>
	<li><a target="__blank" href="https://hla-net.eu/tools/frequency-estimation/">https://hla-net.eu/tools/frequency-estimation/</a></li>
	<li>Santiago Rodriguez, Tom R. Gaunt and Ian N. M. Day. Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies. American Journal of Epidemiology Advance Access published on January 6, 2009, DOI 10.1093/aje/kwn359. http://www.oege.org/software/hwe-mr-calc.html</li>
</ol>
<h4>Further Reading</h4>
Andrews, C. (2010) The Hardy-Weinberg Principle. Nature Education Knowledge 3(10):65 https://www.nature.com/scitable/knowledge/library/the-hardy-weinberg-principle-13235724

Hardy-Weinberg Equilibrium Principle and Genetic Drift - Concept

Hardy-Weinberg und genetische Drift

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are ...

Evolution

Evolutionary Relationships

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system was improved upon during the Renaissance and then, subsequently, by Carolus Linnaeus in the mid 1700&#8217;s. These more formal classification and organization systems grouped species by their physical similarity to one another. For example, all vertebrates have a backbone, but invertebrates do not. Traits like the backbone are called synapomorphies, which are traits that are shared by a group of organisms, presumably because they were derived from a common ancestor. As we will explore, this method has been shown to have limitations and has more recently been amended to include genetic analysis. Still, scientists construct trees called dendrograms to create a visual representation of how species are related to one another and share common ancestors. These dendrograms can aid in our understanding of the evolutionary processes that drive these relationships. Genetic comparisons have added an important tool guiding the analysis of evolutionary relationships.
<h4>Types of Dendrogram</h4>
A type of dendrogram, called a cladogram, depicts the hypothetical genealogical relationships between species with the tips (or leaves) of the tree representing a species and the branches showing how species are related to each other. A slightly more complicated type of tree, called a phylogram, differs from a cladogram in that the branches leading to the species are of different lengths. The length of a branch in this type of tree represents the degree of change between species: the longer the branch, the more time since the species have diverged from a common ancestor. In both types of tree, the common ancestor of a group of species is indicated by a node, which is the point where a series of branches meet. Species that are more closely related to each other (most recently shared a common ancestor) will be located closest to the node. The two species that share a node are called a sister group1.
<h4>Understanding Evolutionary Relationships using Genetic Data</h4>
Historically, cladograms were constructed by comparing the morphology (physical structure) of organisms. This method is still practiced but the techniques have been modernized to include comparison of DNA (deoxyribonucleic acid) sequences between species. Using DNA for building trees has several advantages over relying solely on morphology, including being able to calculate an estimate of how long ago different species shared a common ancestor1. However, using DNA is not always feasible, especially when trees include extinct organisms. DNA is best found in soft tissues, which are not preserved during the fossilization process, and therefore it is uncommon for a DNA sample of an extinct species to be available.
DNA is passed on from parents to their offspring in hereditary units called genes. The nucleotide (A, G, C, and T) sequence of genes found in different species are frequently quite similar, likely due to their having come from a common ancestor. This fact allows researchers to align sequences from different species with one another to build the trees described above. Species with more similarity between their nucleotide sequences will be placed next to each other in a tree, and species with less sequence similarity will be placed further apart from each other.
Bioinformatics are the tools used by biologists to analyze large datasets using a combination of computer science, mathematical modeling, and statistics. One such tool is called BLAST (Basic Local Alignment Search Tool), which can be used to quickly search the entire genome of any species that is available in the NCBI (National Center for Biotechnology Information) database2. The NCBI database combines several different databases that hold different types of DNA sequence information. The process of a BLAST search includes complex computer algorithms, but basically, BLAST aligns sequences of each nucleotide base from a submitted DNA sequence (known as the query sequence) with sequences in the data base that most closely match it. The DNA sequences that are found will be listed in order of similarity to the sequence in question, and will therefore be from species closely related to the species containing the query gene. This comparison may or may not depict the actual evolutionary relationship between species because genes evolve at different rates. Additionally, genomes sometimes contain more than one instance of a similar sequence.
Comparison of DNA sequences of genes is valuable beyond consideration of evolutionary relationships. Frequently, genes are identified in model organisms, such as the fruit fly, Drosophila melanogaster, or the mouse3. Integral to studying a gene, the function of its product is commonly identified and analyzed. If a researcher is interested in studying that function in a different organism (humans for example), BLAST or other bioinformatic tools can be used to find candidate genes based on their similarities to the genes of known function from model organisms.
Human genes can also be used as the starting point to find homologs in model organisms. In fact, human disease research depends heavily on this. Once a human gene of interest is identified, mice can be genetically manipulated to have the homologous gene disrupted, or &#8220;knocked out,&#8221; creating a model of the human disease that can be studied in order to understand and treat the disease. There are many of these mouse strains currently available. For example, there is a mouse model for human Cystic Fibrosis (CF) called the Cftr knockout mouse and another modeling atherosclerosis, called the Apoe knockout3. 
 
<h4>References:</h4>
<ol class="references">
	<li>Annenberg Learner. Online Textbook: Unit 3 Evolution and Phylogenetics. Rediscovering Biology. [Online] 2017. [Cited: August 23, 2018.] https://www.learner.org/courses/biology/textbook/compev/compev_3.html.</li>
	<li>National Center for Biotechnology Information, U.S. National Library of Medicine. BLAST: Basic Local Alignment Search Tool. [Online] https://blast.ncbi.nlm.nih.gov/Blast.cgi.</li>
	<li>National Human Genome Research Institute. Background on Mouse as a Model Organism. National Human Genome Research Institute. [Online] December 2002. [Cited: August 23, 2018.] <a href="https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/" target="__blank">https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/</a>.</li>
</ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Concept

Evolutionäre Beziehungen

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system ...

Animal Diversity

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular eukaryotes that move, consume organic matter, and reproduce sexually. Although these attributes are shared, species within this kingdom are also extremely diverse. This diversity is due to adaptation of each species to a different niche. The niche of a species includes the area, function, and interrelationship of that species with other biotic and abiotic factors in its environment. Niche specialization through evolutionary adaptation allows species to survive and reproduce effectively in their environment and reduces competition among species within the same habitat1-2.
<h4>Classification</h4>
Diversity in Kingdom Animalia has led to the classification of 36 distinct phyla, based on their evolutionary lineage. Seven of these phyla will be discussed, including Porifera, Cnidaria, Platyhelminthes, Annelida, Mollusca, Arthropoda, and Chordata. The first, phylum Porifera, is the earliest, simplest, most ancestral phylum. It includes multicellular, asymmetrical filter feeding sponges that lack distinct tissue layers. Interestingly, sponges can regrow after being broken apart to the level of a single cell. Next, increasing in complexity, the phylum Cnidaria includes jellyfish and coral. Members of this phylum are radially symmetric and diploblastic (possessing two tissue layers). These tissue layers are called the endoderm, which makes up the inner layer, and the ectoderm, which forms the outer layer. Organisms in phylum Cnidaria contain a simple gut with a single opening through which food enters and waste is excreted. Both Porifera and Cnidaria include organisms that are largely sedentary as adults. However, the remaining phyla have evolved adaptations that allow movement and more sophisticated behavior.
Following Cnidaria, there are two phyla of worms, termed Platyhelminthes and Annelida. Worms from both of these phyla breathe by exchanging gases across their skin. As a result, most worms have a large surface area to volume ratio and live in moist or aquatic environments. The skin of these organisms must remain moist to facilitate effective diffusion of gases. Another common characteristic of worms is their bilateral symmetry. This means that an organism can be symmetrically divided in half along a plane. Bilateral symmetry is seen not only in worms, but also in every phyla from this evolutionary point forward. Such symmetry is important to allow directed movement and to concentrate sensory organs near the anterior tip, or head, where they interact with their surroundings. Worms and later phyla also contain an additional tissue layer called the mesoderm that lies between the ectoderm and endoderm. As a result, these organisms are considered &#8220;triploblastic.&#8221;
Of the two phyla of worms, Platyhelminthes is the more ancient and simple. These include flatworms and trematodes, which may be free-living or parasitic. Like Cnidarians, they possess a simple gut with a single opening. Phylum Annelida is more complex, including earthworms, leaches, and polychaeta. These organisms contain a body cavity, called the coelom, between the gut and body wall. They are also distinct due to the segmentation of their bodies. Division of an organism into segments was an important evolutionary step, allowing adaptation of each segment to specialized forms and functions. Major evolutionary advances in this phyla include the development of anterior segments with appendages specialized for grasping and processing food as well as posterior segments with appendages specialized for locomotion.
The next phylum, Mollusca, contains highly morphologically diverse organisms, including octopuses, snails, slugs, and clams. There are few defining features for this group. However, all mollusks possess a mantle, which acts as a protective cover for the respiratory organs, digestive tract, and reproductive structures. Next, the most diverse group of organisms is phylum Arthropoda. This phyla alone makes up approximately three quarters of all living and extinct species known today, including crustaceans, insects, and arachnids. All members of this phylum have a hard, outer coating made of chitin, called an exoskeleton. This exoskeleton is periodically shed for a new, larger one to take its place. Like Annelida, arthropods are segmented. However, instead of multiple repeating sections, arthropods are broken up into three distinct segments called the head, thorax, and abdomen. Most have specialized sensing structures attached to the head called antennae.
Lastly, phylum Chordata includes all animals with a backbone and a few without. Two defining features that unite this group are the presence of a nerve chord and a notochord. The nerve cord is a major part of the chordate nervous system and the notochord is a cartilaginous structure that runs between the nerve cord and the digestive tract. Phylum Chordata includes tunicates (which lose their nerve cord and notochord after metamorphosis from the larval to adult life stage), fish, amphibians, birds, reptiles, and mammals (including humans)3.
<h4>Form and Function</h4>
The vast diversity in body structure, habitats, and behavior observed across all 36 phyla within Kingdom Animalia is truly astounding. To understand these differences better, it is important to consider the connection between form and function. &#8220;Form&#8221; simply means the shape, size, and substance of a structure or organism while &#8220;function&#8221; stands for the way in which that structure is used. Accordingly, the function of a structure can be predicted based on the form observed, or vice versa. For example, a bird specialized to crack hard nuts would be predicted to have a sturdy, sharp beak while a bird that eats fruit may have a smaller, more rounded beak.
Importantly, the form of an organism is constrained by its physiological requirements. For instance, all animals must consume and digest organic matter and exchange gas with their environment. As a result, any adaptations to an animal&#8217;s form must continue to facilitate these processes efficiently. For example, in smaller animals, diffusion of gases across the skin is possible and often useful. However, as the size of an animal increases, its surface area to volume ratio decreases until diffusion across the skin can no longer provide sufficient gas exchange. Such larger animals have developed complex systems to deal with gas exchange, such as the lungs of a whale or the gills of a fish. As a result, the morphology of gas exchange systems depends on both the size of an organism and its environment.
Similarly, it generally follows with other types of adaptations that form follows function, constrained only by the physiological requirement of the organism. However, there are cases in which form does not follow function. Vestigial structures are those that served a purpose in an organism&#8217;s ancestor but are no longer useful in their current form. Such structures have little to no impact on the fitness of an individual, and are thus not evolutionarily selected for or against within the population. As a result, some structures persist with limited functional capacity. For example, the human appendix, a small pocket at the end of the large intestine, is an evolutionary remnant from our ancestors. While this structure has been hypothesized to store beneficial gut bacteria, removal of the appendix is possible with no deleterious effects.
<h4>Convergent Evolution</h4>
Among adaptations seen in Kingdom Animalia, many have developed multiple times throughout evolutionary history. Consider the wings of a bat and those of a bird as an example. These structures are not a result of evolutionary relatedness, but have instead arisen from convergent evolution4. Convergent evolution occurs when two organisms independently evolve similar traits due to similar environmental pressures or niches. The wings of bats, birds, and insets have all developed independently and are said to be &#8220;convergent traits.&#8221; Clearly, such traits are quite beneficial for the niches in which they arise.
<h4>References</h4>
<ol class="references">
	<li>Hollins, J., et al. (2018). 'A physiological perspective on fisheries-induced evolution.' Evol Appl 11(5): 561-576.</li>
	<li>Leong, M., et al. (2017). 'The Habitats Humans Provide: Factors affecting the diversity and composition of arthropods in houses.' Sci Rep 7(1): 15347.</li>
	<li>Wray, G. A. (2015). 'Molecular clocks and the early evolution of metazoan nervous systems.' Philos Trans R Soc Lond B Biol Sci 370(1684).</li>
	<li>Foll, M., et al. (2014). 'Widespread signals of convergent adaptation to high altitude in Asia and America.' Am J Hum Genet 95(4): 394-407.</li>
</ol>

Animal Diversity: Form and Function of Crickets and Crayfish - Concept

Tierische Vielfalt

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular ...

Microbial and Fungal Diversity

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this reason, it is important to understand and distinguish between individual species of these groups. As you will recall, biological taxonomists group organisms based on their phylogenetic relatedness. The three domains of life, Bacteria, Archaea, and Eukaryota segregate life into three distinct groupings. While all bacteria fall within the domain Bacteria, fungi are found in the domain Eukaryota (Kingdom Fungi). In addition to Archaea, domain Bacteria exclusively includes single-celled prokaryotes, meaning their cells lack both a nuclear membrane and membrane-bound organelles. In contrast, fungi of domain Eukaryota have membrane-bound organelles and include both unicellular and multicellular organisms.
<h4>Bacteria</h4>
Bacteria are found nearly everywhere on Earth and are highly adapted to the environments in which they grow. As a result, this domain includes a staggering amount of diversity. The impact of these organisms on human health is equally diverse. For example, species like Streptococcus thermophilus can be used to produce fermented foods like yogurt, while spores of the bacteria Bacillus anthracis are responsible for the potentially deadly disease anthrax.
To aid in the identification of such organism, microbiologist have developed a myriad of tests to characterize both individual cells and entire colonies of bacteria1. One of the most common methods for identifying bacteria is by Gram staining. This test uses colored dyes to determine the composition of the bacteria&#8217;s cell wall. Gram-positive bacteria, which bear a thick cell wall made up of a protein called peptidoglycan, retain a dye called crystal violet and thus appear purple. In contrast, gram-negative bacteria have a thinner cell wall without peptidoglycan and do not retain the crystal violet. Instead, these cells appear pink due to a counter stain called safranin. The use of Gram staining often serves as a preliminary step in the identification of a bacterial species. Further classification requires the identification of additional characteristics. This can include the shape of individual bacteria, which may be either cocci (round), spirillum (spiral), or bacilli (rod-shaped). Additionally, the color, shape, sheen, texture, and even smell of bacterial colonies can be used to distinguish between species.
<h4>Fungi</h4>
Fungi of domain Eukaryota are also highly ecologically diverse and can range in size from microscopic yeast to the largest organism on earth, the honey fungus (Armillaria ostoyae). All fungi are heterotrophic, meaning that they must acquire their food from the environment. These organisms also exhibit diversity in their specific activities, niches, and potential effects on human health. For example, some fungi are consumed or used to produce food by humans while others can cause severe or even deadly infections2.
Within kingdom Fungi, two distinct taxonomic divisions exist based on the reproductive strategy of the species. First, Ascomycota, known as the sac fungi, have a reproductive structure called an ascus. Together, many asci form the ascocarp, or &#8220;fruiting body&#8221; of the fungi. These fungi can reproduce sexually or asexually by budding. Ascomycota consist of some economically important species like the yeast used in brewing beer and baking bread, and some species popular in cuisine, such as morels and truffles. The second group, Basidiomycota, includes organisms commonly known as &#8220;club fungi&#8221; due to the presence of a large club-shaped reproductive structure called the basidium. In contrast to Ascomycota, most Basidomucota reproduce exclusively by sexual reproduction. This occurs when two haploid mycelia join to form a basidiocarp. On a button mushroom, this is the cap structure. On the underside of the basidiocarp, reproductive structures called gills lined with structures called basidia produce haploid nuclei that eventually form basidiospores. Basidiospores are then dispersed by the wind. This taxonomic division includes many commonly eaten button mushrooms, as well as wild puff mushrooms.
<h4>Importance of Microbiology</h4>
Overall, it is clear that the diverse functions and properties of microorganisms make their identification extremely important for human health and well-being. While a vast number of species of fungi and bacteria have been identified to date, it is estimated that millions more have yet to be identified and classified. In addition, human disciplines that explore Fungi and Bacteria are as diverse as the organisms themselves and will undoubtedly continue to grow. The relevance of microorganisms to the medical fields is more prevalent now then ever, as multi-antibiotic resistant species of infectious bacteria threaten human health. Understanding the structure and biological characteristics of infectious and non-infectious species provides key insights necessary for the creation of new antibiotic treatments and preventative strategies3-4.
Microbiology is also critical to the food industry. Numerous products, including yogurt, cheese, kombucha, bread, and alcohol are made using specific categories or species of microbes. At the same time, food scientists focus on engineering chemical compounds and preservatives that will reduce the growth of unwanted microbes on food. In all these processes, an understanding of how microbes survive, reproduce, and affect their environment is necessary.
<h4>References </h4>
<ol class="references">
	<li>Hiroshi Asakura, Holger Brueggemann, Sou-ichi Makino, and Yoshiko Sugita-Konishi, &#8220;Molecular Approaches for the Classification of Microbial Pathogens of Public Health Significance,&#8221; BioMed Research International, vol. 2014, Article ID 725801, 2 pages, 2014. https://doi-org.bdigitaluss.remotexs.co/10.1155/2014/725801.</li>
	<li>Treseder, K. K. and J. T. Lennon (2015). 'Fungal traits that drive ecosystem dynamics on land.' Microbiol Mol Biol Rev 79(2): 243-262.</li>
	<li>Arvanitis, M. and E. Mylonakis (2015). 'Fungal-bacterial interactions and their relevance in health.' Cell Microbiol 17(10): 1442-1446.</li>
	<li>Aarestrup, F. M., et al. (2008). 'Resistance in bacteria of the food chain: epidemiology and control strategies.' Expert Rev Anti Infect Ther 6(5): 733-750.</li>
</ol>

Microbial Colony and Fungal Diversity - Concept

Mikrobielle und Pilzvielfalt

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this ...

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Preparation of Onion Root Tips and Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">For the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water to grow roots for several days. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, clean the onion roots of any dirt or debris. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, in a 1.5 mL tube, dissolve 10 mcg of nocodazole per 1 mL of dimethyl sulfoxide solution, making 1 tube per student. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, set out all of the other tools, materials, and reagents from the list of materials onto each student&#39;s lab bench. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Prep

Zellteilung

Preparation of Onion Root Tips and Solutions
ExpandFor the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water ...

Zellprozesse

Lab Manual Sub Articles

Bacterial Transformation

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Bacterial Transformation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">First, ensure that each student or student group has a filled ice bucket.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, set up a tube rack for each work station.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Into the tube racks, place two empty, sterile 1.5 mL tubes, and then two one mL aliquots of SOC media.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, place the plasmid tube into the ice bucket and add two tubes of competent E. coli cells, allowing them to thaw if frozen.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set out two LB agar plates per group and two LB agar ampicillin plates per group.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Ensure the students have the appropriate pipettes.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">And finally, set a water bath to 42 &#176;C and a temperature-controlled shaker to 37 &#176;C.
</div></li></ul></li></ol>

Bacterial Transformation using Plasmids - Prep

Bakterielle Transformation

Bacterial Transformation
ExpandFirst, ensure that each student or student group has a filled ice bucket.
Then, set up a tube rack for each work station.
...

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Cheek Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Immediately before the experiment, wash and peel onion bulbs for the class.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Remove the entire brown outer skin and cut the onion in half with a knife. Pull apart the layers of the onion. The thin, nearly transparent film layers within the onion will be used by the students.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the onion film into a Petri dish and then place this into the refrigerator until the students arrive.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Bring out the microscopes and set them on the lab bench.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set the microscopes to the lowest magnification and plug them into an electrical outlet.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Clean the lenses with lens paper or laboratory wipes and test the knobs to make sure the lenses focus properly.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Make sure that the stage can be moved.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Prep

Zellstruktur

Visualizing Onion and Cheek Cells
ExpandImmediately before the experiment, wash and peel onion bulbs for the class.
Remove the entire brown outer skin and ...