Name: phenotypic characterization of macrophages from rat kidney by flow cytometry
Uploaded: 2016-10-18T14:00:00.000+00:00
Duration: 7 min 14 s
Description: Watch this Scientific Journal Video about Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry at JoVE.com

This work was supported by grants from FIS/FEDER (Programa Miguel Servet: CP10/00479, PI13/00802 and PI14/00883), Spanish Society of Atherosclerosis, Spanish Society of Nephrology and Fundaci&#242;n Renal I&#241;igo Alvarez de Toledo (FRIAT) to Juan Antonio Moreno. FIS/FEDER funds PI14/00386 and Instituto Reina Sof&#236;a de Investigaci&#242;n Nefrol&#242;gica to Jes&#250;s Egido. Fundaci&#242;n Conchita Rabago to Melania Guerrero Hue. Fundaci&#242;n Renal I&#241;igo Alvarez de Toledo (FRIAT) to Alfonso Rubio Navarro.

该指令欧洲议会2010/63 / EU和国家指南二千零十三分之五十三下面这个协议被批准由当地机构动物护理和使用委员会。 1.试剂和溶液的制备<ol><li>制备所有的试剂和溶液在无菌条件下并在层流罩下使用。保持在4℃下的解决方案。 </li><li>制备染色缓冲液（2％胎牛血清（FBS）的在1×Dulbecco氏PBS）中。 </li><li>加入0.5毫克胶原酶每个毫升盐水的制备胶原酶溶液。 </li><li>制备氯胺酮/赛拉嗪的麻醉溶液（2:1体积/体积）。 </li></ol> 2.肾脏灌注和提取<ol><li>氯胺酮/甲苯噻嗪腹腔注射（75毫克/ 12毫克/千克体重）麻醉大鼠。仔细捏皮肤的小折，以检查该动物被充分麻醉。然后，盖上眼睛兽医药膏，以防止干燥时的麻醉下。注:4-纹个大的Wistar大鼠在此测定法中使用。 </li><li>一旦大鼠被完全麻醉，将其放置在手术台上以仰卧姿势。 </li><li>应用70％的乙醇于腹部。 </li><li>做一个切口，中央通过腹部皮肤和腹膜，从耻骨到胸腔，暴露胸腔和腹腔。 </li><li>注入盐溶液（0.9％）为腹主动脉灌注直到所有血液从肾脏除去使用灌注系统肾脏。切开主动脉在腹部的水平，以帮助释放血液。 </li><li>从肾门（肾静脉，动脉和输尿管）切割取下大鼠双侧肾脏。为了解封肾脏，用手指，小心地分离胶囊11按下肾脏的边缘。 </li><li>放置肾成Hanks&#39;平衡盐溶液中。 </li></ol> 3.肾消化细胞悬液<ol><li>切半肾用剪刀切成小块，并把片放入1.5 ml管。 注:以下所有浓度为1个样品（1/2大鼠的肾脏）计算。 </li><li>加入1 ml的胶原酶溶液中，并在37℃孵育30分钟。通过颠倒混合，每5分钟，以确保该胶原酶溶液可将整个组织。 </li><li>收集溶液，并使之通过一个过滤器（40微米）配有一个柱塞的帮助，并且在10毫升染色缓冲液重悬它。 </li><li>离心在400×g离心15分钟。 </li><li>重悬在1ml的ACK（氯化铵 - 钾）溶解缓冲液沉淀。 1分钟在室温下30秒后，加入10 mL染色缓冲液以停止反应。 </li><li>离心在400×g离心10分钟。 </li><li>重悬在染色缓冲液（1ml）中的适当体积的沉淀并用过滤器（30微米）过滤细胞悬浮液。 </li><li>计数上使用血球台盼蓝排除细胞的数目和2百万个细胞添加到1.5ml管站都进不去。 </li></ol> 4.细胞染色和流式细胞仪分析<ol><li>离心细胞，在100×g离心5分钟。 </li><li>重悬在100μl大鼠血清的粒料（稀释1:100），10分钟，在4℃下阻断Fc受体。 </li><li>通过加入1ml染色缓冲液和离心机100 xg的5分钟洗涤。 </li><li>混合为细胞表面染色的抗体在100μl染色缓冲液为每个条件:CD45 APC-Cy7的（1:100），CD163 A647（4:100）和溶液来检测活细胞（3:1000）。 </li><li>在黑暗中再悬浮在抗体混合物20分钟将细胞沉淀，在4℃。 </li><li>通过加入1ml染色缓冲液和离心机在100×g离心5分钟洗涤。重复此步骤。 </li><li>在4°C黑暗环境中添加600微升固定/通透的解决方案20分钟。 </li><li>在170 xg离心用1ml透/洗涤缓冲液，离心洗涤2次，每次5分钟。 </li><li>添加CD68 FITC抗体（35:1000）为intracel细胞性染色至100μl透/洗涤缓冲液对各条件。 </li><li>重悬在CD68抗体溶液中的沉淀，并在黑暗中在4℃孵育50分钟。 </li><li>用1ml透/洗涤缓冲液，离心洗涤，在170×g离心5分钟。 </li><li>重悬在100μl透/洗涤缓冲液的沉淀，添加CD86PE抗体（35:1000），并在黑暗中在4℃孵育20分钟。 </li><li>通过加入1ml透/洗涤的，在100×g离心5分钟，缓冲液和离心清洗。 </li><li>重悬在100μl透/洗涤缓冲液的颗粒和把它传递给流式细胞管的流动。 </li><li>通过流式细胞仪分析样品。决定在侧向散射光/前向散射光（SSC / FSC）窗门控活细胞CD45染色。在第二步骤中，分析在CD68 +细胞的CD86和CD163表达。 </li></ol>

<ol>
	<li>Gansevoort, R. T., 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=Chronic+kidney+disease+and+cardiovascular+risk:+epidemiology,+mechanisms,+and+prevention.">Chronic kidney disease and cardiovascular risk: epidemiology, mechanisms, and prevention.</a> Lancet. 382, 339-352 (2013).</li><li>Kon, V., Linton, M. F., Fazio, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atherosclerosis+in+chronic+kidney+disease:+the+role+of+macrophages.">Atherosclerosis in chronic kidney disease: the role of macrophages.</a> Nat. Rev. Nephrol. 7, 45-54 (2011).</li><li>Kim, J. H., 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=Macrophage+depletion+ameliorates+glycerol-induced+acute+kidney+injury+in+mice.">Macrophage depletion ameliorates glycerol-induced acute kidney injury in mice.</a> Nephron Exp. Nephrol. 128, 21-29 (2014).</li><li>Belliere, J., 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=Specific+macrophage+subtypes+influence+the+progression+of+rhabdomyolysis-induced+kidney+injury.">Specific macrophage subtypes influence the progression of rhabdomyolysis-induced kidney injury.</a> J. Am. Soc. Nephrol. 26, 1363-1377 (2015).</li><li>Kinsey, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+dynamics+in+AKI+to+CKD+progression.">Macrophage dynamics in AKI to CKD progression.</a> J. Am. Soc. Nephrol. 25, 209-211 (2014).</li><li>Lech, M., 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=Macrophage+phenotype+controls+long-term+AKI+outcomes--kidney+regeneration+versus+atrophy.">Macrophage phenotype controls long-term AKI outcomes--kidney regeneration versus atrophy.</a> J. Am. Soc. Nephrol. 25, 292-304 (2014).</li><li>Murray, P. J., 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=Macrophage+activation+and+polarization:+nomenclature+and+experimental+guidelines.">Macrophage activation and polarization: nomenclature and experimental guidelines.</a> Immunity. 41, 14-20 (2014).</li><li>Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages.">Alternative activation of macrophages.</a> Nat. Rev. Immunol. 3, 23-35 (2003).</li><li>Mosser, D. M., Edwards, 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=Exploring+the+full+spectrum+of+macrophage+activation.">Exploring the full spectrum of macrophage activation.</a> Nat. Rev. Immunol. 8, 958-969 (2008).</li><li>Ortiz, A., 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=Translational+value+of+animal+models+of+kidney+failure.">Translational value of animal models of kidney failure.</a> Eur. J. Pharmacol. 759, 205-220 (2015).</li><li>Martina, M. N., Bandapalle, S., Rabb, H., Hamad, A. R. <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+double+negative+alphabeta+T+cells+from+the.">Isolation of double negative alphabeta T cells from the.</a> J. Vis. Exp. , (2014).</li><li>Martin-Fernandez, B., 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=Aldosterone+Induces+Renal+Fibrosis+and+Inflammatory+M1-Macrophage+Subtype+via+Mineralocorticoid+Receptor+in+Rats.">Aldosterone Induces Renal Fibrosis and Inflammatory M1-Macrophage Subtype via Mineralocorticoid Receptor in Rats.</a> PLoS. One. 11, e0145946 (2016).</li><li>Layoun, A., Samba, M., Santos, M. M. <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+murine+peritoneal+macrophages+to+carry+out+gene+expression+analysis+upon+Toll-like+receptors+stimulation.">Isolation of murine peritoneal macrophages to carry out gene expression analysis upon Toll-like receptors stimulation.</a> J. Vis. Exp. (e52749), (2015).</li><li>Komohara, Y., 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=Macrophage+infiltration+and+its+prognostic+relevance+in+clear+cell+renal+cell+carcinoma.">Macrophage infiltration and its prognostic relevance in clear cell renal cell carcinoma.</a> Cancer Sci. 102, 1424-1431 (2011).</li><li>Han, Y., Ma, F. Y., Tesch, G. H., Manthey, C. L., Nikolic-Paterson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+macrophages+in+the+fibrotic+phase+of+rat+crescentic+glomerulonephritis.">Role of macrophages in the fibrotic phase of rat crescentic glomerulonephritis.</a> Am. J. Physiol Renal Physiol. 304, F1043-F1053 (2013).</li><li>Ndisang, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+heme+oxygenase-adiponectin-atrial+natriuretic+peptide+axis+in+renal+function.">Role of the heme oxygenase-adiponectin-atrial natriuretic peptide axis in renal function.</a> Curr. Pharm. Des. 21, 4380-4391 (2015).</li><li>Blackbeard, J., 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=Quantification+of+the+rat+spinal+microglial+response+to+peripheral+nerve+injury+as+revealed+by+immunohistochemical+image+analysis+and+flow+cytometry.">Quantification of the rat spinal microglial response to peripheral nerve injury as revealed by immunohistochemical image analysis and flow cytometry.</a> J. Neurosci. Methods. 164, 207-217 (2007).</li><li>Strobl, H., Scheinecker, C., Csmarits, B., Majdic, O., Knapp, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+of+intracellular+CD68+molecule+expression+in+normal+and+malignant+haemopoiesis.">Flow cytometric analysis of intracellular CD68 molecule expression in normal and malignant haemopoiesis.</a> Br. J. Haematol. 90, 774-782 (1995).</li></ol>

巨噬细胞是发挥不同炎性疾病，包括肾疾病中起重要作用的异构细胞。有在肾脏疾病中的巨噬细胞子集的表征越来越大的兴趣，因为每个细胞巨噬细胞亚群以不同的方式对肾损伤的发展有助于，据报道在肾小球肾炎，糖尿病性肾病和肾癌14-16。在急性肾损伤的早期阶段中，M1巨噬细胞的一个优势是观察，促进肾小管坏死和炎症。然而，在稍后阶段的M2巨噬细胞有较高含量被观察到的解决炎症并参与组织重塑。在慢性肾病，M1和M2的巨噬细胞同时共存在，尽管M1可以是主要的，从而增加了与永久炎性肾损害。因此，重要的是要提高我们的巨噬细胞亚型在肾脏病的病理生理作用的知识。免疫组化研究不能确定在一个可靠的方法17的巨噬细胞亚群的百分比。因此，有必要开发新的技术，以定量地确定不同的巨噬细胞的表型，并了解这些细胞对肾的炎症反应的作用。 该原稿描述的协议通过流式细胞术来确定在大鼠肾脏的巨噬细胞子集的数量和表型。在我们的研究中，大鼠进行醛固酮施用肾损害的炎性模型。在此模型中，我们观察到增加的 CD45 +和C68 +巨噬细胞浸润如通过流式细胞术确定，并通过免疫组织化学证实。此外，M1巨噬细胞（CD68 + / CD86 +），但不M2巨噬细胞的富集，分别在醛固酮处理的小鼠中观察到。这些结果由确定M 1（iNOS和IFN-γ）的mRNA表达和M2巨噬进一步证实年龄标记（的Arg1或IL-10）。隔离协议期间流式细胞仪分析报低细胞死亡率。有几个因素会影响细胞生存力和表面标记检测，例如组织离解的程度以及持续时间和组织消化（酶促，机械等 ）的类型。过度消化增加细胞死亡和巨噬细胞活化，并降低表面标记的表达，而相反的效果可能会从低肾脏离解的结果。然而，当我们的协议在从大鼠腹膜分离巨噬细胞进行的，在这些细胞中观察到CD86和CD163的增强的存在。 我们的协议已被优化，以确定从大鼠醛固酮介导的高血压在肾脏细胞巨噬细胞的异质性，并且因此，可以被外推到其它炎性肾疾病模型。但是，此过程可适于每种实验条件，因为炎症修改组织的完整性，从而影响组织的消化胶原酶率。 要注意的是用于测定总的巨噬细胞含量是很重要的，将细胞固定和透化以允许与CD68抗体18内标记。对于固定的协议包括使用甲醛的可能影响荧光染料缀合的初级抗体，尤其是藻红蛋白。要解决这个问题，除了固定/通透的解决方案，并进一步CD68染色后进行孵育CD86-PE抗体。另一方面，需要注意的是甲醛也可以与可能后的巨噬细胞分离执行的其他测定法，如mRNA表达测定干扰是很重要的。不过，也有可从甲醛固定细胞分离的mRNA商业试剂盒。 总之，该协议描述了肾的巨噬细胞的表型特征的方法在一个quantitati已经使用流式细胞仪的方式。总体而言，这里描述的方案是易于使用，可重复的，和有用的从大鼠肾脏区分不同巨噬细胞群体。

Renal disease is a global health problem, with increased prevalence, and associated with elevated morbidity and mortality1. One of the most important mechanisms involved in the progression and development of renal injury is inflammation, mainly triggered by macrophages. Macrophages play a pivotal role in many inflammatory diseases, including renal disorders2. Thus, an elevated presence of infiltrating macrophages has been reported in biopsies from patients with acute kidney injury (AKI) or chronic kidney disease (CKD)3,4. Recent studies suggest that the long-term outcome of renal disease could be controlled by macrophages5,6. In response to the local microenvironment, macrophages may differentiate into different phenotypes that play diverse biological functions7. Two well differentiated macrophage phenotypes have been established: classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2)8. M1 macrophages promote inflammation, whereas M2 macrophages have an anti-inflammatory role and are involved in tissue repair9. Therefore, a better knowledge of macrophage heterogeneity is necessary to understand their regulation and contribution to renal pathology and develop novel therapeutic approaches. 
Both, murine and rats models have been widely used to understand the molecular and cellular mechanism involved in renal injury10. However, there are substantial differences in the diverse markers used to identify macrophages phenotypes between these rodents. Hence, several murine markers, such as F4/80 or Ly6C are not used in rats, thus limiting the extrapolation of findings between these species. Moreover, there is a limited number of markers describing macrophage phenotypes in rats, explaining the scarce studies analyzing macrophage heterogeneity in these animals as compared with mice. Therefore, new strategies for macrophage subset characterization are necessary to understand the role of macrophages in renal disease models in rats. 
This manuscript describes a protocol for the phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. This technique can be further followed by several assays, including cell sorting and mRNA or protein expression studies to allow in-depth characterization of the role of macrophages in renal disease.

<table><tbody><tr><td>Laminar flow hood</td><td>Faster</td><td></td><td>Or equivalent equipment</td></tr><tr><td>Centrifuge</td><td>Hettich</td><td></td><td>Or equivalent equipment</td></tr><tr><td>Flow cytometer (FACSAria)</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>BioWest</td><td>S1820-500</td><td></td></tr><tr><td>PBS 10x</td><td>LONZA</td><td>BE17-515Q</td><td></td></tr><tr><td>Collagenase</td><td>Sigma-Aldrich</td><td>12/1/9001</td><td></td></tr><tr><td>ACK Lysing Buffer</td><td>Thermo Fisher Scientific</td><td>A10492-01</td><td></td></tr><tr><td>Flow cytometry strainers</td><td>BD Biosciences</td><td>340626</td><td></td></tr><tr><td>Falcon cell strainers</td><td>Thermo Fisher Scientific</td><td>352340</td><td></td></tr><tr><td>Flow cytometry tubes</td><td>Falcon</td><td>352052</td><td>5 ml Polystyrene Round-Bottom Tube</td></tr><tr><td>Centrifuge tubes</td><td>Corning centristar</td><td>430791</td><td></td></tr><tr><td>Water bath</td><td>Memmert GmbH + Co. KG</td><td>WNE 7</td><td>37 &amp;#176;C</td></tr><tr><td>Fixation/Permeabilization Solution or Permeabilization/Wash Buffer</td><td>BD Biosciences</td><td>554714</td><td></td></tr><tr><td>Rompum (Xylazine)</td><td>Bayer</td><td></td><td>Or equivalent</td></tr><tr><td>Ketalar (Ketamine)</td><td>Pfizer</td><td></td><td>Or equivalent</td></tr><tr><td>Hanks&amp;rsquo; balanced salt solution</td><td>Sigma-Aldrich</td><td>H8264-500ML</td><td></td></tr><tr><td>Saline solution</td><td>Braun</td><td>622415</td><td></td></tr><tr><td>Anti-CD45 (clone:OX-1) APC-Cy7</td><td>Biolegend</td><td>202216</td><td>Diluted 1:100</td></tr><tr><td>Anti-CD68 (clone: ED1) FITC</td><td>Bio-RAD</td><td>MCA341F</td><td>Diluted 35:1,000</td></tr><tr><td>anti-CD86 (clone: 24F) PE</td><td>Biolegend</td><td>200307</td><td>Diluted 35:1,000</td></tr><tr><td>anti-CD163 (clone: ED2) Alexa Fluor 647</td><td>Bio-RAD</td><td>MCA342R</td><td>Diluted 4:100</td></tr><tr><td>Live/dead stain&amp;nbsp;</td><td>Molecular Probes</td><td>L34955</td><td>Diluted 3:1,000</td></tr></tbody></table>

phenotypic characterization of macrophages from rat kidney by flow cytometry

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.

我们分析了巨噬细胞的异质性与肾脏巨噬细胞浸润的存在增加相关肾损伤的炎症实验模型。在此模型中，肾损伤在饮水中Wistar大鼠3周诱导醛固酮（1毫克-1千克-1天）加的盐（NaCl 1％）的施用，如先前报道12。 我们的实验中的时间表中示出在图1中，首先，肾细胞中分离，采用机械方法和用胶原酶（ 图1A）进一步酶消化。此后，红细胞裂解并通过使用流式细胞仪检测肾巨噬细胞。细胞用抗CD45，CD86和CD163为膜检测和CD68细胞内染色（ 图1B）一起温育。最后，巨噬细胞p的表型根据方案opulations分析显示出图1C。 肾细胞巨噬细胞含量通过与CD45和CD68（ 图2A）双阳性选择细胞进行分析。使用上述协议，增加在醛固酮处理的大鼠中观察到的 CD45 + / CD68 +巨噬细胞相比，对照组（0.57±0.16对0.25±0.02，％CD45 + / CD68每总肾细胞+巨噬细胞）（ 图2，表见插图 ）。 的 CD45 + / CD68 +巨噬细胞的细胞活力，如通过流式细胞术用紫染料染色测定，是常规大于95％。此后，CD86（M1标记物）和CD163（M2标记物）的表达中所评估的（数据未示出） CD45 + / CD68 +细胞。醛固酮增多行政CD45 + / CD68 + / CD86 + M1的含量巨噬细胞（2.87±2.3对1.36±0.68;％ 的 CD45 + / CD68 + / CD86 +每总CD45 + / CD68 +细胞的巨噬细胞）（ 图2）。然而，在这两个醛固酮治疗组和对照组中观察到的 CD45 + / CD68的低编号+ / CD163 + M2巨噬细胞。来分析低CD163表达是否被实验方案中涉及到的蛋白水解脱落，我们重复此过程，包括胶原酶消化，在来自大鼠腹膜分离的巨噬细胞，如前所述13。 如图2B报道，CD45 + / CD68 +巨噬细胞为阳性既CD86和CD163，验证所选择的抗体的效用，并确认我们的协议为M1 / M2巨噬细胞的大鼠肾脏表征的有效性。 为了验证这些结果，与上述实验模型的肾损伤相关的炎症反应通过免疫组织化学和实时PCR分析。如在图3报道，增加在醛固酮+盐处理的大鼠中观察到CD68 +巨噬细胞，与对照组相比。然后，进行了测定巨噬细胞的表型标记物来表征肾M1 / M2巨噬细胞分布。醛固酮+盐处理后观察到的iNOS增加mRNA的表达和IFN-γ（M1标记），而的Arg1或IL-10的mRNA表达量（M2巨噬细胞标记）醛固酮给药后没有改变。总之，这些结果证实，醛固酮+盐给药介导的体内的炎性M1巨噬细胞的表型。 <img alt="图1" src="/files/ftp_upload/54599/54599fig1.jpg" /> 图1:协议肾细胞的分离和巨噬细胞流式细胞术检测。 </stron肾细胞隔离（A）和巨噬细胞的检测用流式细胞仪（B）的g&gt;的示意图。根据不同的分布CD45，CD68，CD86和CD163标志（C）不同的巨噬细胞表型的代表方案。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54599/54599fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54599/54599fig2.jpg" /> 图2:M1和M2巨噬细胞标志物在肾脏从醛固酮通过流式细胞仪时大鼠 CD45染色的代表点图在侧向散射光/前向散射光（SSC / FSC）窗口（ 门控活细胞， 离开了 。 ）从在控制和醛固酮+盐处理的动物的肾脏悬浮液。在散点图内的框标识活细胞表达CD45。包括在这个盒子细胞进行分析以确定CD68 +细胞CD86和CD163表达（A，右 ）。散点图腹膜的巨噬细胞作为用于检测CD45，CD68，CD86和CD163的阳性对照，采用先前所用的相同的策略。该数据以平均值±SEM（B）。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54599/54599fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54599/54599fig3.jpg" /> 图3:醛固酮+盐务管理局后炎症反应。 （A）CD68染色和的代表性图像（B）中通过RT-PCR对M1标记（iNOS和INFγ）和M2标记（的Arg1和IL-10）的控制和醛固酮+盐处理的大鼠测量mRNA水平的定量分析。数据速递ED平均±SEM。 * P &lt;0.05与对照。比例尺:100微米<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54599/54599fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry at JoVE.com

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

该原稿描述的表型，并通过流式细胞从大鼠肾脏定居巨噬细胞的定量分析的详细协议。所得染色的细胞也可以用于其它应用，包括细胞分选，基因表达分析或功能性研究，从而增加在实验模型中获得的信息。

通过流式细胞仪大鼠肾脏巨噬细胞的表型特征

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

phenotypic-characterization-macrophages-from-rat-kidney-flow

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

14.7K 次观看.  Autonoma University. 该原稿描述的表型，并通过流式细胞从大鼠肾脏定居巨噬细胞的定量分析的详细协议。所得染色的细胞也可以用于其它应用，包括细胞分选，基因表达分析或功能性研究，从而增加在实验模型中获得的信息。 

视频: Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

科研

免疫与感染

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

收获小鼠肺泡巨噬细胞和评价聚酸酐纳米粒子诱导的细胞活化

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

生物工程

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

抑制接枝反应性免疫的调节性巨噬细胞的功能表征

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Reliable and High Efficiency Extraction of Kidney Immune Cells

Immune system activation occurs in multiple kidney diseases and pathophysiological processes. The immune system consists of both adaptive and innate components and multiple cell types. Sometimes, the cell type of interest is present in very low numbers among the large numbers of total cells isolated from the kidney. Hence, reliable and efficient isolation of kidney mononuclear cell populations is important in order to study the immunological problems associated with kidney diseases. Traditionally, tissue isolation of kidney mononuclear cells have been performed via enzymatic digestions using different varieties and strengths of collagenases/DNAses yielding varying numbers of viable immune cells. Recently, with the development of the mechanical tissue disruptors for single cell isolation, the collagenase digestion step is avoided and replaced by a simple mechanical disruption of the kidneys after extraction from the mouse. Herein, we demonstrate a simple yet efficient method for the isolation of kidney mononuclear cells for every day immune cell extractions. We further demonstrate an example of subset analysis of immune cells in the kidney. Importantly, this technique can be adapted to other soft and non-fibrous tissues such as the liver and brain.

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this goal.

肾脏免疫细胞的可靠和高效率的提取

Immune system activation occurs in multiple kidney diseases and pathophysiological processes.  The immune system consists of both adaptive and innate ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

通过流式细胞术分析来自中枢神经系统的小胶质细胞和单核细胞衍生的巨噬细胞

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To Fc&#947;R Cross-linking

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in response to various immune stimuli. ROS generated during immune activation exerts potent antimicrobial activity primarily through the ability of ROS to damage DNA and proteins, causing death of microorganisms. Being able to measure ROS production reproducibly and with ease is necessary in order to assess the contribution of various pathways and molecules to this mechanism of host defense. In this paper, we demonstrate the use of fluorescent probes and flow cytometry to detect ROS production. Although widely used, fluorescent measurement of ROS is notoriously problematic, especially with regards to measurement of ROS induced by specific and not mitogenic stimuli. We present a detailed methodology to detect ROS generated as a result of specific Fc&#947;R stimulation beginning with macrophage generation, priming, staining, Fc&#947;R cross-linking, and ending with flow cytometric analysis.

This study demonstrates the use of flow cytometry to detect reactive oxygen species (ROS) production resulting from activation of the Fc&#947;R. This method can be used to assess changes in the antimicrobial and redox signaling function of phagocytes in response to immune complexes, opsonized microorganisms, or direct Fc&#947;R cross-linking.

巨噬细胞 ros 产生的流式细胞仪测量对 fcγr 交联的响应

The oxidative or respiratory burst is used to describe the rapid consumption of oxygen and generation of reactive oxygen species (ROS) by phagocytes in ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

应用流式细胞仪对人体脂肪组织免疫细胞的表征

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

医学

Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. Innate immune cells, specifically mononuclear phagocytes, express overlapping cell surface markers, which makes identifying these populations a challenge. Multi-parameter flow cytometry allows for the simultaneous, quantitative analysis of multiple cell surface markers in order to differentiate monocytes, macrophages, microglia, and dendritic cells in mouse eyes. This protocol describes the enucleation of whole mouse eyes, ocular dissection, digestion into a single cell suspension, and staining of the single cell suspension for myeloid cell markers. Additionally, we explain the proper methods for determining voltages using single color controls and for delineating positive gates using fluorescence minus one controls. The major limitation of multi-parameter flow cytometry is the absence of tissue architecture. This limitation can be overcome by multi-parameter flow cytometry of individual ocular compartments or complimentary immunofluorescence staining. However, immunofluorescence is limited by its lack of quantitative analysis and reduced number of fluorophores on most microscopes. We describe the use of multi-parametric flow cytometry to provide highly quantitative analysis of mononuclear phagocytes in laser-induced choroidal neovascularization. Additionally, multi-parameter flow cytometry can be used for the identification of macrophage subsets, fate mapping, and cell sorting for transcriptomic or proteomic studies.

This protocol provides a method to digest whole eyes into a single cell suspension for the purpose of multi-parameter flow cytometric analysis in order to identify specific ocular mononuclear phagocytic populations, including monocytes, microglia, macrophages, and dendritic cells.

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

The innate immune system plays important roles in ocular pathophysiology including uveitis, diabetic retinopathy, and age-related macular degeneration. ...

Flow Cytometry Approach to Characterize Phagocytic Properties of Acutely-Isolated Adult Microglia and Brain Macrophages In Vitro

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent A&#946; fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Assessment of phagocytic properties of microglia and brain macrophages can provide a valuable functional dimension to molecular profiling studies. We describe a validated protocol that uses flow cytometry to rapidly and reliably quantify phagocytosis of fluorescent microspheres and amyloid beta fibrils by acutely-isolated microglia and brain macrophages from mouse models.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in ...

神经科学

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

从人内脏脂肪组织中分离出活的脂肪细胞和基质血管组分，适用于 RNA 分析和巨噬细胞表型分析

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...