Name: synchronization of caulobacter crescentus for investigation of the bacterial cell cycle
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Description: Watch this Scientific Journal Video about Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle at JoVE.com

The authors thank members of the Shapiro lab and Erin Schrader for comments on the manuscript. The authors acknowledge financial support from: NIH postdoctoral fellowship F32 GM100732 to JMS and NIH grants R01 GM51426 and R01 GM32506 to LS.

1. على نطاق واسع التزامن - الأمثل لالبقعة الغربية، ميكروأري / RNA تسلسل، وغيره فحوصات مكثفة مواد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> من مخزون الثلاجة أو لوحة، وتنمو 5 مل O / N ثقافة سلالة NA1000 عن طريق هز في 28 ° C في PYE المتوسطة. </li><li style=";text-align:right;direction:rtl"> تطعيم 0.5 مل من الخلايا من الخطوة 1 في 25 مل من M2G (الجداول 1-2) ويهز عند 28 درجة مئوية حتى تصل الثقافة إلى OD 600 بين 0.5 و 0.6. </li><li style=";text-align:right;direction:rtl"> تطعيم الخلايا في 1 لتر من M2G ويهز في 28 ° C. </li><li style=";text-align:right;direction:rtl"> مرة واحدة OD 600 يصل 0،5-0،6، تأكد من وجود مخابئ الخلايا باستخدام السائل شنت المرحلة المجهري. بقعة 1 ميكرولتر من الخلايا على شريحة زجاجية، وتغطي مع انزلاق الغطاء، وصورة من المرحلة المجهري. تأكيد وجود خلايا مخابئ من خلال وضع تصور خلايا السباحة بسرعة بين السكان. </li><li style=";text-align:right;direction:rtl"> تدور الخلايا لمدة 15 دقيقة في 7 kxg في 4 درجات مئوية في الدوار JA-10. </li><li style=";text-align:right;direction:rtl"> تجاهل طاف وإضافة 180 مل من M2 البارد (الجداول 1-2) والدقة بلطفuspend جميع الخلايا باستخدام الماصة المصلية. تجاهل الخلايا مكعبات فضفاضة. فهي خلايا predivisional ومطاردة. </li><li style=";text-align:right;direction:rtl"> إضافة 60 مل من البرد حل السيليكا الغروية (تأكد من خلط السيليكا تعليق صمغي جيدا قبل إضافة إلى الخلايا)، ومزيج تعليق خلية أيضا. </li><li style=";text-align:right;direction:rtl"> صب تعليق خلية في ثمانية 30 مل أنابيب وتدور لمدة 30 دقيقة في 6.4 kxg في 4 درجات مئوية في JA-20 الدوار. ملاحظة: يجب للمرء أن يرى شريطين مميزة؛ الفرقة مخابئ هي أقل الفرقة بينما مطاردة / خلايا predivisional هي في أعلى الفرقة (الشكل 1B). </li><li style=";text-align:right;direction:rtl"> نضح بعناية الفرقة أعلى قبالة وإزالة السائل إلى ~ 1 سم فوق الفرقة مخابئ (أقل من الفرقة). </li><li style=";text-align:right;direction:rtl"> (الحرجة) باستخدام الماصة باستور، وإزالة بعناية الفرقة مخابئ ووضع في أنبوب نظيفة. ليغسل السيليكا الغروية، أعلى الأنبوب قبالة مع M2 البارد وتدور لمدة 10 دقيقة في 6.4 kxg في 4 درجات مئوية في JA-20 الدوار. </li><li style=";text-align:right;direction:rtl"> تجاهل بعناية طاف و resuspendالخلايا في 20 مل من M2 البارد وتدور لمدة 10 دقيقة في 6.4 kxg في 4 درجات مئوية في JA-20 الدوار. </li><li style=";text-align:right;direction:rtl"> و resuspend جميع الكريات في 30 مل من M2 البارد وقياس OD 600 باستخدام مقياس الطيف الضوئي وفارغة باستخدام البارد المتوسطة M2. حفظ 1 ميكرولتر للمرحلة التصوير للتحقق من مخابئ الخلايا. يجب أن يكون 90-95٪ من خلايا swarmers. </li><li style=";text-align:right;direction:rtl"> تدور باستمرار خلايا لمدة 5 دقائق عند 6.4 kxg في 4 درجات مئوية في JA-20 الدوار. Resuspend الخلايا في 28 ° C M2G المتوسطة بحيث A 600 غير ~ 0.3-0.4 والبدء اهتزاز في 28 ° C. ملاحظة: غلة النموذجية هي ما بين 30 و 60ML من مخابئ ثقافة خلية من خلايا 1L غير المتزامنة. </li><li style=";text-align:right;direction:rtl"> البدء في اتخاذ نقاط زمنية (لن ​​ثقافة نوع البرية تأخذ حوالي 135-140 دقائق لتقسيم) 8،9. في كل نقطة زمنية، وقياس OD 600. تأكد من أن OD 600 بعد تقسيم ما يقرب من 2X وOD الأولي 600. </li><li style=";text-align:right;direction:rtl"> لالغربية لطخة أو التعبير الجيني المقايسات، وإزالة مأخوذة من 1mL من مكعبlture عند نقاط الوقت المطلوب، وتدور إلى أسفل في أقصى سرعة في جهاز للطرد المركزي الطاولة لمدة 30 ثانية، وبسرعة صب أو نضح المتوسط، وفلاش تجميد بيليه خلية في النيتروجين السائل. تخزين الخلايا عند -80 درجة مئوية حتى تحليل المصب. </li></ol> 2. على نطاق صغير التزامن - الأمثل للالمجهر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> من مخزون الثلاجة أو لوحة، وتنمو 5 مل O / N الثقافة تهتز عند 28 درجة مئوية في M2G. </li><li style=";text-align:right;direction:rtl"> تمييع في 15 مل من M2G (الجداول 1-2) وتنمو حتى سجل منتصف (OD 600 = 0.5-0.6). </li><li style=";text-align:right;direction:rtl"> تدور في 6.4 kxg لمدة 10 دقيقة في 4 درجات مئوية في JA-20 الدوار، وresuspend في 1 مل الباردة M2 (الجداول 1-2) ونقل إلى أنبوب microcentrifuge 2 مل. </li><li style=";text-align:right;direction:rtl"> تدور في 15 kxg لمدة 3 دقائق في أنبوب microcentrifuge لتكوير الخلايا، ونضح قبالة طاف، ووضع بيليه على الجليد، وresuspend في 900 ميكرولتر من M2 البارد. </li><li style=";text-align:right;direction:rtl"> إضافة 900 ميكرولتر من PVP البارد المغلفة السيليكا الغروية وتدور لمدة 20 دقيقة في 15 kxg في 4 درجات مئوية في هيئة التصنيع العسكريأنبوب rocentrifuge. </li><li style=";text-align:right;direction:rtl"> (الناقد) نضح أو الماصة قبالة رأس مطاردة / predivisional الفرقة الخلية وجمع أسفل مخابئ الفرقة في أنبوب microcentrifuge جديد. </li><li style=";text-align:right;direction:rtl"> غسل الخلايا مخابئ مرتين في 1 مل من M2 البارد في حين الطرد المركزي في 15 kxg لمدة 3 دقائق. </li><li style=";text-align:right;direction:rtl"> قبل تدور النهائي، نقل الخلايا في قبل المبردة 1 مل اختبار أنبوب زجاجي وقياس OD 600 من الخلايا مقارنة فارغة من M2. </li><li style=";text-align:right;direction:rtl"> resuspend الكرية خلية النهائي إلى 28 ° C M2G في لOD بين 0،3-0،4 ويهز خلية / لفة في 28 ° C. ملاحظة: غلة النموذجية هي ما بين 2 و 4 مل من ثقافة الخلية مخابئ. </li><li style=";text-align:right;direction:rtl"> للتجارب المجهري، في مكان المطلوب نقاط زمنية 1 ميكرولتر من الخلايا على لوحة agarose M2G للتصوير. </li></ol>

<ol>
	<li>McAdams, H. H., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=System-level+design+of+bacterial+cell+cycle+control.">System-level design of bacterial cell cycle control.</a> FEBS Lett. 583, 3984-3991 (2009).</li><li>McAdams, H. H., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+architecture+and+conservation+pattern+of+whole-cell+control+circuitry.">The architecture and conservation pattern of whole-cell control circuitry.</a> J. Mol. Biol. 409, 28-35 (2011).</li><li>McAdams, H. H., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bacterial+cell-cycle+regulatory+network+operating+in+time+and+space.">A bacterial cell-cycle regulatory network operating in time and space.</a> Science. 301, 1874-1877 (2003).</li><li>Ptacin, J. L., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiating+bacterial+mitosis:+understanding+the+mechanism+of+ParA-mediated+chromosome+segregation.">Initiating bacterial mitosis: understanding the mechanism of ParA-mediated chromosome segregation.</a> Cell Cycle. 9, 4033-4034 (2010).</li><li>Evinger, M., Agabian, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Envelope-associated+nucleoid+from+Caulobacter+crescentus+stalked+and+swarmer+cells.">Envelope-associated nucleoid from Caulobacter crescentus stalked and swarmer cells.</a> J. Bacteriol. 132, 294-301 (1977).</li><li>Tsai, J. W., Alley, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+of+the+Caulobacter+McpA+chemoreceptor+is+cell+cycle+regulated+by+a+ClpX-dependent+pathway.">Proteolysis of the Caulobacter McpA chemoreceptor is cell cycle regulated by a ClpX-dependent pathway.</a> J. Bacteriol. 183, 5001-5007 (2001).</li><li>Marks, M. E., 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=The+genetic+basis+of+laboratory+adaptation+in+Caulobacter+crescentus.">The genetic basis of laboratory adaptation in Caulobacter crescentus.</a> J. Bacteriol. 192, 3678-3688 (2010).</li><li>Ely, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+Caulobacter+crescentus.">Genetics of Caulobacter crescentus.</a> Methods Enzymol. 204, 372-384 (1991).</li><li>Williams, B., Bhat, N., Chien, P., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ClpXP+and+ClpAP+proteolytic+activity+on+divisome+substrates+is+differentially+regulated+following+the+Caulobacter+asymmetric+cell+division.">ClpXP and ClpAP proteolytic activity on divisome substrates is differentially regulated following the Caulobacter asymmetric cell division.</a> Mol. Microbiol. 93, 853-866 (2014).</li><li>Quon, K. C., Yang, B., Domian, I. J., Shapiro, L., Marczynski, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Negative+control+of+bacterial+DNA+replication+by+a+cell+cycle+regulatory+protein+that+binds+at+the+chromosome+origin.">Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin.</a> Proc. Natl. Acad. Sci. U. S. A. 95, 120-125 (1998).</li><li>Laub, M. T., Chen, S. L., Shapiro, L., McAdams, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+directly+controlled+by+CtrA,+a+master+regulator+of+the+Caulobacter+cell+cycle.">Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle.</a> Proc. Natl. Acad. Sci. U. S. A. 99, 4632-4637 (2002).</li><li>Quon, K. C., Marczynski, G. T., Shapiro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+control+by+an+essential+bacterial+two-component+signal+transduction+protein.">Cell cycle control by an essential bacterial two-component signal transduction protein.</a> Cell. 84, 83-93 (1996).</li><li>Ferullo, D. J., Cooper, D. L., Moore, H. R., Lovett, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+synchronization+of+Escherichia+coli+using+the+stringent+response,+with+fluorescence+labeling+assays+for+DNA+content+and+replication.">Cell cycle synchronization of Escherichia coli using the stringent response, with fluorescence labeling assays for DNA content and replication.</a> Methods. 48, 8-13 (2009).</li><li>Degnen, S. T., Newton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+replication+during+development+in+Caulobacter+crescentus.">Chromosome replication during development in Caulobacter crescentus.</a> J. Mol. Biol. 64, 671-680 (1972).</li><li>Bates, D., 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=The+Escherichia+coli+baby+cell+column:+a+novel+cell+synchronization+method+provides+new+insight+into+the+bacterial+cell+cycle.">The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle.</a> Mol. Microbiol. 57, 380-391 (2005).</li><li>Abel, 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=Bi-modal+distribution+of+the+second+messenger+c-di-GMP+controls+cell+fate+and+asymmetry+during+the+caulobacter+cell+cycle.">Bi-modal distribution of the second messenger c-di-GMP controls cell fate and asymmetry during the caulobacter cell cycle.</a> PLoS Genet. 9, e1003744 (2013).</li><li>Johnson, R. C., Ely, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+spontaneously+derived+mutants+of+Caulobacter+crescentus.">Isolation of spontaneously derived mutants of Caulobacter crescentus.</a> Genetics. 86, 25-32 (1977).</li><li>Britos, L., 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=Regulatory+response+to+carbon+starvation+in+Caulobacter+crescentus.">Regulatory response to carbon starvation in Caulobacter crescentus.</a> PLoS One. 6, e18179 (2011).</li><li>Boutte, C. C., Crosson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complex+logic+of+stringent+response+regulation+in+Caulobacter+crescentus:+starvation+signalling+in+an+oligotrophic+environment.">The complex logic of stringent response regulation in Caulobacter crescentus: starvation signalling in an oligotrophic environment.</a> Mol. Microbiol. 80, 695-714 (2011).</li></ol>

The authors have nothing to disclose.

The bacterial cell cycle is a fundamental process in life and is important for the study of growth and as a target for next generation antibiotics. Here, we detailed the rapid synchronization procedures for C. crescentus NA1000, a model organism for the study of the bacterial cell cycle and asymmetric cell division. This method is amendable to western blot, gene expression profiling, and fluorescence microscopy assays to investigate the spatiotemporal regulation of the bacterial cell cycle. 
The protocol is quick and yields healthy synchronized cells. Other synchronization methods require long starvation of cells and rely on the stringent response to arrest the cell cycle in the same state, leading to cells that are dissimilar to cultures in logarithmic growth 13. While &#8220;baby machines&#8221; and other surface attachment methods have been used successfully14,15, they typically have low yields of synchronized cells. The density centrifugation protocols presented here allow higher purity and yields of synchronized C. crescentus NA1000 swarmer cells.
Additional complications can lead to potential problems throughout the synchrony procedure. Have a liquid mount phase microscope handy to optically check the purity of cells before synchronization and to check the purity of swarmers. A good synchrony will yield >95% swarmer cells. Check the OD600 on a spectrophotometer to ensure the cells are doubling during the time course of the synchrony. 
Cells don&#8217;t synchronize as well in the presence of antibiotics or some mutations. If the strain does not carry a replicating plasmid, try to remove the antibiotics from the growth medium to improve swarmer cell yield. Cells grown in the presence of antibiotics or mutant strains will also have a slower cell cycle, so it is important to check the division time by microscopy for comparison to healthy strains. 
Avoid vortexing the cells, as this will shear off the flagella and lower the quality of the synchrony. In preparation for the synchrony protocol don&#8217;t grow the cells above OD600 0.5. This ensures healthy growth that isn&#8217;t altered by high cell density. Be careful when aspirating/pipetting off supernatants containing the stalked and predivisional cell populations. In particular, do not to touch the swarmer band which may lead to contamination and/or a loss in yield of the swarmer cells. The cell pellet will be very loose in the first wash of cells after removing them from the Colloidal silica. Be careful when removing the supernatant not to disrupt the pellet.
Occasionally NA1000 cells can lose synchronization capacity due to a loss of DNA in the prophage region of the genome 7. In this case, no swarmer band is observed or the swarmer band intensity is dramatically reduced. If possible, collect cells if present in the swarmer band and streak onto a PYE plate to re-isolate the synchronizable cells. If working with a mutant strain, it is often useful to reintroduce the mutation into a fresh batch of NA1000. Conversely, mutation of critical cell cycle regulatory proteins can, in some cases, disrupt the ability of cells to synchronize16.
M2G medium was initially described with a higher 20 mM phosphate concentration 8,17; however, current studies use a lower 10 mM concentration as presented in Table 1 18,19. Alternative to the standard M2G medium, it is possible to use richer PYE medium. Here the unsynchronized growth and growth after synchronization can be substituted for PYE.
C. crescentus NA1000 provides a valuable experimental tool to study the bacterial cell cycle. Additionally, due to the single copy of the chromosome in the swarmer cell, this bacterium has also become a powerful model to study the structure of the bacterial chromosome.

تسيطر على دورة الخلية البكتيرية كل من تكرار الجينوم وتقسيم الخلايا الوليدة. الأهم من ذلك، كما مقاومة المضادات الحيوية تشكل تهديدا متزايدا على الصحة العامة، تقدم دورة الخلية البكتيرية هدفا غير مستغلة للتنمية المضادات الحيوية. في بكتيريا العنيقاء crescentus، كل دورة الخلية يؤدي إلى تقسيم غير المتماثلة، مما أسفر عن خليتين ابنة مصائر مختلفة (الشكل 1A) 1،2. خلية ابنة واحدة يرث السوط وهي متحركة في حين أن ابنة أخرى يرث ساق وهي اطئة. دائرة الوراثية متكاملة تسيطر تقدم دورة الخلية ومصير الخلية عن طريق تنظيم النسخي، الفوسفات، مما يشير، والتحلل البروتيني المنظم 3. وبالإضافة إلى ذلك، وتكرار كروموسوم وخلايا ابنة العائد الفصل المتزامنة التي تحتوي بالضبط نسخة واحدة من كروموسوم 4. الأهم من ذلك، هذه أنواع الخلايا اثنين يمكن فصلها بسرعة عن طريق كولوايدال السيليكا الجسيمات كثافة الطرد المركزي في سلالة synchronizable NA1000 5-7 السماح للعزل الخلايا مخابئ عن بقية السكان مع عوائد عالية (الشكل 1B). عزل مخابئ خلايا ثم المضي قدما بشكل متزامن من خلال انقسام الخلايا غير المتماثلة. هنا، ونحن بالتفصيل بروتوكول يستخدم لمزامنة العنيقاء سلالة NA1000. ونحن نقدم البروتوكولات ونصائح استكشاف الأخطاء وإصلاحها المشتركة لكلا الكبيرة والصغيرة الحجم تزامن. يوفر هذا الإجراء التجريبي أداة قوية لاستجواب السيطرة الزمانية المكانية من دورة الخلية العنيقاء ومصير الخلية.

<table><tbody><tr><td>PVP Coated Colloidal Silica (Percoll)</td><td>Sigma-Aldrich</td><td>P4937</td><td></td></tr><tr><td>Colloidal Silica (Ludox AS-40)</td><td>Sigma-Aldrich</td><td>420840</td><td></td></tr><tr><td>JA10 Rotor</td><td>Beckman-Coulter</td><td>369687</td><td></td></tr><tr><td>JA20 Rotor</td><td>Beckman-Coulter</td><td>334831</td><td></td></tr><tr><td>Ferrous Sulfate Chelate Solution</td><td>Sigma-Aldrich</td><td>F0518</td><td></td></tr><tr><td>30 ml Centrifuge Tubes</td><td>Corning</td><td>8445</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD</td><td>SX0720-1</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>VWR</td><td>BDH9268-500G</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Amresco</td><td>0621-500g</td><td></td></tr></tbody></table>

synchronization of caulobacter crescentus for investigation of the bacterial cell cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

تزامن عادة ينتج شريطين من الخلايا (الشكل 1B): الفرقة مخابئ، والتي لديها أعلى كثافة، ومطاردة / predivisional الفرقة خلية من أقل كثافة. لضمان وتشمل الضوابط المشتركة تزامن كفاءة مراقبة OD 600 وقياس مستويات البروتين CTRA التي كتبها طخة غربية في نقاط متميزة الوقت دورة الخلية. وOD 600 يجب زيادة بنحو 2 أضعاف خلال دورة الخلية (الشكل 2). لطخة غربية لدورة الخلية منظم سيد CTRA هو السيطرة مفيدة للتحقق من التزامن جيد (الشكل 2). ويستخدم CTRA في الخلية مخابئ لمنع تكرار الحمض النووي وتتحلل عند بداية تكرار الحمض النووي (10). CTRA ثم يتم تصنيعه في وقت لاحق في دورة الخلية وينشط النسخ من مجموعة من الجينات التنموية بما في ذلك العديد من مكونات السوط 11،12. وسوف يكون ناجحا التزامن رله نمط تتأرجح مستويات البروتين CTRA. <img alt="الشكل (1)" src="/files/ftp_upload/52633/52633fig1.jpg" /> الشكل 1. دورة الخلية من العنيقاء crescentus. (A) التخطيطي الكرتون من دورة الخلية مخابئ. الخلايا مخابئ تفرق في تكرار خلايا مطاردة المختصة التي كتبها التراجع بيلي، إخراج سياط، والشروع في تكرار الحمض النووي. الدوائر وثيتا الهياكل معروضة داخل الخطوط العريضة خلية تمثل الكروموسومات هادئة وتكرار. خلايا ثم التقدم من خلال دورة الخلية بناء السوط واحد في القطب قبالة ساق. بناء على التقسيم، يتم إنشاء نوعين خلية فريدة من نوعها، منعت النسخ المتماثل متحركة مخابئ خلية وخلية ثابتة المختصة تكرار مطاردة. (ب) ممثل النتائج لكثافة الطرد المركزي. انخفاض الكثافة احقت وpredivخلايا isional تطفو بالقرب من أعلى التدرج في حين كثيفة مخابئ الخلايا في نهاية المطاف باتجاه الجزء السفلي من الأنبوب. الحانات النطاق هي 1 ميكرون في صور المجهر المرحلة. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig1large.jpg" target="_blank">الرجاء انقر هنا لمشاهدة نسخة أكبر من هذا الرقم.</a> <img alt="الشكل 2" src="/files/ftp_upload/52633/52633fig2.jpg" /> الشكل 2. ممثل النتائج لنجاح مخابئ التزامن الخلية. وكتلة الخلية يقاس OD 600 ينبغي أن تزيد ببطء ويتضاعف في نهاية المطاف طوال فترة الفحص. تم pipetted 1 مل مأخوذة في cuvettes البلاستيكية من التزامن خلية كبيرة وOD 600 قياسها باستخدام مقياس الطيف الضوئي في النقاط الزمنية المشار إليها. بالإضافة إلى ذلك، ينبغي أن تكون دورة الخلية سيد البروتين التنظيمي CTRA الحالي في الخلية مخابئ لمنع تكرار الحمض النووي، تليها التدهور السريعتتزامن مع بدء تكرار الحمض النووي. CTRA يتم بعد ذلك إعادة إحياء لاحقا في دورة الخلية لتنشيط التعبير عن العديد من الجينات التنموية بما في ذلك مكونات سوطي وبيلي حرجة. أجريت البقع الغربية لدورة الخلية سيد البروتين التنظيمي CTRA من خلال اتخاذ 1 مل مأخوذة من التزامن على نطاق واسع. ومعلق الخلايا في 250 ميكرولتر Laemmlli عينة العازلة في OD 600، فصل على 10٪ تريس، الغليسين PAGE، ونقل إلى PVDF، ونشف مع anti-CTRA الأجسام المضادة. تؤدي إجراءات التزامن فشل في CTRA البقع الغربية مع أي تغيير في مستويات البروتين. وقد حضنت α-CTRA الأجسام المضادة 12 في 1: 10،000 التخفيف عن 1.5 ساعة في 3٪ الحليب TBST وغسلها 3 مرات في TBST. غسلها 10،000 التخفيف في 3٪ الحليب TBST لمدة 1 ساعة، 3 مرات مع TBST، وتصوير الفيلم على استخدام عدة كشف chemiluminescent: الماعز α-أرنب الثانوي ثم أضاف في 1. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52633/52633fig2large.jpg" target="_blank">من فضلك</a>انقر هنا لمشاهدة نسخة أكبر من هذا الرقم. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> نا 2 هبو 4 </td><td> 17.4 ز </td></tr><tr><td> KH 2 PO 4 </td><td> 10.6 ز </td></tr><tr><td> NH 4 الكلورين </td><td> 10 ز </td></tr><tr><td> H 2 O </td><td> resuspend في 1 L &amp; الأوتوكلاف </td></tr></tbody></table> الجدول 1. 20X M2 أملاح وصفة. <table border="0" cellpadding="0" cellspacing="0" style=";text-align:right;direction:rtl"><tbody><tr><td> 20X M2 أملاح </td><td> 50 مل </td><td></td></tr><tr><td> 0.5 M MgSO 4 </td><td> 1 مل </td><td></td></tr><tr><td> 20٪ الجلوكوز </td><td> 10 مل </td><td> بديلا عن H 2 O في M2 </td></tr><tr><td> كبريتات الحديدوز كلات الحل </td><td> 1 مل </td><td></td></tr><tr><td> 0.1 M CaCl 2 </td><td> 5 مل </td><td> إضافة آخر من AVهطول الأمطار OID </td></tr><tr><td> H 2 O </td><td> ملء تصل إلى 1 L </td><td></td></tr><tr><td> تصفية العقيمة مع مرشح 0.22 ميكرومتر </td><td></td><td></td></tr></tbody></table> الجدول 2. M2 وM2G صفة.

Watch this Scientific Journal Video about Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle at JoVE.com

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

تزامن<em&gt; العنيقاء Crescentus</em&gt; للتحقيق في دورة الخلية البكتيرية

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

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Biology

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

11.8K Views.  Stanford University School of Medicine. Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

Video: Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

دراسة الخلية التعبير عن تنظيم دورة الجينات من قبل اثنين من بروتوكولات التزامن الخلية التكميلية

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Genetics

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

تعيش خلية من التصوير<emالعصوية الرقيقة> و العقدية الرئوية الآلي باستخدام الميكروسكوب مرور الزمن

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Immunology and Infection

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes underlying central cell cycle events. However, imaging of these molecules in slow-growing bacteria represents a challenge due to photobleaching of fluorophores and phototoxicity during image acquisition. Here, we describe a simple protocol to circumvent these limitations in the case of Myxococcus xanthus (which has a generation time of 4 - 6 h). To this end, M. xanthus cells are grown on a thick nutrient-containing agar pad in a temperature-controlled humid environment. Under these conditions, we determine the doubling time of individual cells by following the growth of single cells. Moreover, key cellular processes such as chromosome segregation and cell division can be imaged by fluorescence live-cell imaging of cells containing relevant fluorescently labeled marker proteins such as ParB-YFP, FtsZ-GFP, and mCherry-PomX over multiple cell cycles. Subsequently, the acquired images are processed to generate montages and/or movies.

Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

Bacterial cells are spatially highly organized. To follow this organization over time in slow growing Myxococcus xanthus cells, a set-up for fluorescence live-cell imaging with high spatiotemporal resolution over several generations was developed. Using this method, spatiotemporal dynamics of important proteins for chromosome segregation and cell division could be determined.

الأسفار خلايا حية تصوير "دورة الخلية الخضري كاملة" من "بكتيريا الاجتماعية" بطيئة النمو إكسانثوس ميكسوكوككوس

Fluorescence live-cell imaging of bacterial cells is a key method in the analysis of the spatial and temporal dynamics of proteins and chromosomes ...

Biochemistry

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

القياس المستمر للضوضاء البيولوجية في الإشريكية القولونية باستخدام المجهر الفاصل الزمني

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Bioengineering

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

المجهرية الخلوية التفاعلية للتحقيق في توطين البروتين تحت الخلوي والتغيرات في الخلايا المورفولوجية في البكتيريا

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Temporal Tracking of Cell Cycle Progression Using Flow Cytometry without the Need for Synchronization

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell synchronization can be difficult for many cell systems. Standard practice is to block cell cycle progression at a specific stage and then release the accumulated cells producing a wave of cells progressing through the cycle in unison. However, some cell types find this block toxic resulting in abnormal cell cycling, or even mass death. Bromodeoxyuridine (BrdU) uptake can be used to track the cell cycle stage of individual cells. Cells incorporate this synthetic thymidine analog, while synthesizing new DNA during S phase. By providing BrdU for a brief period it is possible to mark a pool of cells that were in S phase while the BrdU was present. These cells can then be tracked through the remainder of the cell cycle and into the next round of replication, permitting the duration of the cell cycle phases to be determined without the need to induce a potentially toxic cell cycle block. It is also possible to determine and correlate the expression of both internal and external proteins during subsequent stages of the cell cycle. These can be used to further refine the assignment of cell cycle stage or assess effects on other cellular functions such as checkpoint activation or cell death.

This protocol describes the use of bromodeoxyuridine (BrdU) uptake to permit the temporal tracking of cells that were in S phase at a specific point in time. Addition of DNA dyes and antibody labeling facilitates detailed analysis of the fate of the S phase cells at later times.

تتبع الزمني للتقدم دورة الخلية باستخدام التدفق الخلوي دون الحاجة إلى مزامنة

This protocol describes a method to permit the tracking of cells through the cell cycle without requiring the cells to be synchronized. Achieving cell ...

Live Cell Fluorescence Microscopy to Observe Essential Processes During Microbial Cell Growth

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of proteins which are essential for bacterial survival. A series of target-specific dyes can be used as probes to better understand these processes. Staining with lipophilic dyes enables the observation of membrane structure, visualization of lipid microdomains, and detection of membrane blebs. Use of fluorescent-d-amino acids (FDAAs) to probe the sites of peptidoglycan biosynthesis can indicate potential defects in cell wall biogenesis or cell growth patterning. Finally, nucleic acid stains can indicate possible defects in DNA replication or chromosome segregation. Cyanine DNA stains label living cells and are suitable for time-lapse microscopy enabling real-time observations of nucleoid morphology during cell growth. Protocols for cell labeling can be applied to protein depletion mutants to identify defects in membrane structure, cell wall biogenesis, or chromosome segregation. Furthermore, time-lapse microscopy can be used to monitor morphological changes as an essential protein is removed and can provide additional insights into protein function. For example, the depletion of essential cell division proteins results in filamentation or branching, whereas the depletion of cell growth proteins may cause cells to become shorter or rounder. Here, protocols for cell growth, target-specific labeling, and time-lapse microscopy are provided for the bacterial plant pathogen Agrobacterium tumefaciens. Together, target-specific dyes and time-lapse microscopy enable characterization of essential processes in A. tumefaciens. Finally, the protocols provided can be readily modified to probe essential processes in other bacteria.

Understanding the function of essential processes in bacteria is challenging. Fluorescence microscopy with target-specific dyes can provide key insights into microbial cell growth and cell cycle progression. Here, Agrobacterium tumefaciens is used as a model bacterium to highlight methods for live cell imaging for characterization of essential processes.

يعيش خلية Fluorescence مجهرية لمراقبة العمليات الأساسية أثناء نمو الخلايا الميكروبية

Core cellular processes such as DNA replication and segregation, protein synthesis, cell wall biosynthesis, and cell division rely on the function of ...

Combining Mitotic Cell Synchronization and High Resolution Confocal Microscopy to Study the Role of Multifunctional Cell Cycle Proteins During Mitosis

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The synchronization of cell populations at specific stages of the cell cycle has been found to be very useful in such experimental endeavors. Synchronization of cells by treatment with chemicals that are relatively less toxic can be advantageous over the use of pharmacological inhibitory drugs for the study of consequent cell cycle events and to obtain specific enrichment of selected mitotic stages. Here, we describe the protocol for synchronizing human cells at different stages of the cell cycle, including both in S phase and M phase with a double thymidine block and release procedure for studying the functionality of mitotic proteins in chromosome alignment and segregation. This protocol has been extremely useful for studying the mitotic roles of multifunctional proteins which possess established interphase functions. In our case, the mitotic role of Cdt1, a protein critical for replication origin licensing in G1 phase, can be studied effectively only when G2/M-specific Cdt1 can be depleted. We describe the detailed protocol for depletion of G2/M-specific Cdt1 using double thymidine synchronization. We also explain the protocol of cell fixation, and live cell imaging using high resolution confocal microscopy after thymidine release. The method is also useful for analyzing the function of mitotic proteins under both physiological and perturbed conditions such as for Hec1, a component of the Ndc80 complex, as it enables one to obtain large sample sizes of mitotic cells for fixed and live cell analysis as we show here.&#160;&#160;

We present a protocol for double thymidine synchronization of HeLa cells followed by analysis using high resolution confocal microscopy. This method is key to obtaining large number of cells that proceed synchronously from S phase to mitosis, enabling studies on mitotic roles of multifunctional proteins which also possess interphase functions.

الجمع بين الخلية الانقسامية المزامنة وعالية الدقة [كنفوكل] الفحص المجهري لدراسة دور البروتينات المتعددة الوظائف دورة الخلية أثناء الانقسام

Study of the various regulatory events of the cell cycle in a phase-dependent manner provides a clear understanding about cell growth and division. The ...

Single-cell Microfluidic Analysis of Bacillus subtilis

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers single-cell resolution and long observation times spanning hundreds of generations; unlike agarose pad-based microscopy, it has uniform growth conditions that can be tightly controlled. Because the continuous flow of growth medium isolates the cells in a microfluidic device from unpredictable variations in the local chemical environment caused by cell growth and metabolism, authentic changes in gene expression and cell growth in response to specific stimuli can be more confidently observed. Bacillus subtilis is used here as a model bacterial species to demonstrate a &#34;mother machine&#34;-type method for cellular analysis. We show how to construct and plumb a microfluidic device, load it with cells, initiate microscopic imaging, and expose cells to a stimulus by switching from one growth medium to another. A stress-responsive reporter is used as an example to reveal the type of data that may be obtained by this method. We also briefly discuss further applications of this method for other types of experiments, such as analysis of bacterial sporulation.

Single-cell Microfluidic Analysis of Bacillus subtilis

We present a method for the microfluidic analysis of individual bacterial cell lineages using Bacillus subtilis as an example. The method overcomes shortcomings of traditional analytical methods in microbiology by allowing observation of hundreds of cell generations under tightly controllable and uniform growth conditions.

تحليل موائع جزيئية خلية واحدة نجحت عصية

Microfluidic technology overcomes many of the limitations to traditional analytical methods in microbiology. Unlike bulk-culture methods, it offers ...

Laser Micro-Irradiation to Study DNA Recruitment During S Phase

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both endogenous and exogenous sources such as metabolic activities or UV radiation. Without DNA repair, the cell's genetic code becomes compromised, undermining the structures and functions of proteins and potentially causing disease. 
Understanding the spatiotemporal dynamics of the different DNA repair pathways in various cell cycle phases is crucial in the field of DNA damage repair. Current fluorescent microscopy techniques provide great tools to measure the recruitment kinetics of different repair proteins after DNA damage induction. DNA synthesis during the S phase of the cell cycle is a peculiar point in cell fate regarding DNA repair. It provides a unique window to screen the entire genome for mistakes. At the same time, DNA synthesis errors also pose a threat to DNA integrity that is not encountered in non-dividing cells. Therefore, DNA repair processes differ significantly in S phase as compared to other phases of the cell cycle, and those differences are poorly understood. 
The following protocol describes the preparation of cell lines and the measurement of dynamics of DNA repair proteins in S phase at locally induced DNA damage sites, using a laser-scanning confocal microscope equipped with a 405 nm laser line. Tagged PCNA (with mPlum) is used as a cell cycle marker combined with an AcGFP-labeled repair protein of interest (i.e., EXO1b) to measure the DNA damage recruitment in S phase.

This protocol describes a non-invasive method to efficiently identify S-phase cells for downstream microscopy studies, such as measuring DNA repair protein recruitment by laser micro-irradiation.

الليزر الإشعاع الدقيق لدراسة تجنيد الحمض النووي خلال مرحلة S

DNA damage repair maintains the genetic integrity of cells in a highly reactive environment. Cells may accumulate various types of DNA damage due to both ...

تزامن

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