Name: digestion of whole mouse eyes for multi-parameter flow cytometric analysis of mononuclear phagocytes
Uploaded: 2020-06-17T14:45:00
Description: Watch this Scientific Journal Video about Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes at JoVE.com

日航得到NIH赠款K08EY030923的支持：CMC得到了来自NIH国家关节炎和肌肉骨骼疾病研究所的K01赠款（5K001AR060169）和来自狼疮研究联盟的小说研究补助金（637405）的支持。

所有程序均由西北大学机构动物护理和使用委员会批准。C57BL/6小鼠被分组安置在西北大学比较医学中心的屏障设施中。芝加哥，伊利诺伊州）。所有动物都被安置在12/12小时的光/暗循环中，可以免费获得食物和水。
1. 眼组织集合
<ol><li>牺牲10-12周大的C57BL/6J小鼠使用受管制的二氧化碳管理，直到小鼠反应迟钝，不再吸入。执行宫颈错位或其他批准的次要方法，以确保安乐死?...

<ol>
	<li>Chinnery, H. R., McMenamin, P. G., Dando, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+physiology+in+the+eye.">Macrophage physiology in the eye.</a> Pflugers Archiv: European Journal of Physiology. 469 (3-4), 501-515 (2017).</li><li>Droho, S., Cuda, C. M., Perlman, H., Lavine, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-Derived+Macrophages+Are+Necessary+for+Beta-Adrenergic+Receptor-Driven+Choroidal+Neovascularization+Inhibition.">Monocyte-Derived Macrophages Are Necessary for Beta-Adrenergic Receptor-Driven Choroidal Neovascularization Inhibition.</a> Investigative Ophthalmology &amp; Visual Science. 60 (15), 5059-5069 (2019).</li><li>O'Koren, E. G., 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=Microglial+Function+Is+Distinct+in+Different+Anatomical+Locations+during+Retinal+Homeostasis+and+Degeneration.">Microglial Function Is Distinct in Different Anatomical Locations during Retinal Homeostasis and Degeneration.</a> Immunity. 50 (7), 723-727 (2019).</li><li>Kratofil, R. M., Kubes, P., Deniset, 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=Monocyte+Conversion+During+Inflammation+and+Injury.">Monocyte Conversion During Inflammation and Injury.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 37 (1), 35-42 (2017).</li><li>Zimmermann, H. W., Trautwein, C., Tacke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+role+of+monocytes+and+macrophages+for+the+inflammatory+response+in+acute+liver+injury.">Functional role of monocytes and macrophages for the inflammatory response in acute liver injury.</a> Frontiers in Physiology. 3, 56 (2012).</li><li>Gautier, E. 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=Gene-expression+profiles+and+transcriptional+regulatory+pathways+that+underlie+the+identity+and+diversity+of+mouse+tissue+macrophages.">Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages.</a> Nature Immunology. 13 (11), 1118-1128 (2012).</li><li>Sutti, 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=CX3CR1+Mediates+the+Development+of+Monocyte-Derived+Dendritic+Cells+during+Hepatic+Inflammation.">CX3CR1 Mediates the Development of Monocyte-Derived Dendritic Cells during Hepatic Inflammation.</a> Cells. 8 (9), 1099 (2019).</li><li>Liu, 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=Myeloid+Cells+Expressing+VEGF+and+Arginase-1+Following+Uptake+of+Damaged+Retinal+Pigment+Epithelium+Suggests+Potential+Mechanism+That+Drives+the+Onset+of+Choroidal+Angiogenesis+in+Mice.">Myeloid Cells Expressing VEGF and Arginase-1 Following Uptake of Damaged Retinal Pigment Epithelium Suggests Potential Mechanism That Drives the Onset of Choroidal Angiogenesis in Mice.</a> PLoS One. 8 (8), 72935 (2013).</li><li>Satpathy, A. 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=Zbtb46+expression+distinguishes+classical+dendritic+cells+and+their+committed+progenitors+from+other+immune+lineages.">Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages.</a> The Journal of Experimental Medicine. 209 (6), 1135-1152 (2012).</li><li>Krause, T. A., Alex, A. F., Engel, D. R., Kurts, C., Eter, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF-Production+by+CCR2-Dependent+Macrophages+Contributes+to+Laser-Induced+Choroidal+Neovascularization.">VEGF-Production by CCR2-Dependent Macrophages Contributes to Laser-Induced Choroidal Neovascularization.</a> PLoS One. 9 (4), 94313 (2014).</li><li>Lin, W., Liu, T., Wang, B., Bi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ocular+dendritic+cells+in+uveitis.">The role of ocular dendritic cells in uveitis.</a> Immunology Letters. 209, 4-10 (2019).</li><li>Ginhoux, F., Schultze, J. L., Murray, P. J., Ochando, J., Biswas, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+multidimensional+concept+of+macrophage+ontogeny,+activation+and+function.">New insights into the multidimensional concept of macrophage ontogeny, activation and function.</a> Nature Immunology. 17 (1), 34-40 (2016).</li><li>O'Koren, E. G., Mathew, R., Saban, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+reveals+that+microglia+and+recruited+monocyte-derived+macrophages+are+definitively+distinguishable+by+phenotype+in+the+retina.">Fate mapping reveals that microglia and recruited monocyte-derived macrophages are definitively distinguishable by phenotype in the retina.</a> Science Reports. 6, 20636 (2016).</li><li>Bennett, M. 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=New+tools+for+studying+microglia+in+the+mouse+and+human+CNS.">New tools for studying microglia in the mouse and human CNS.</a> Proceedings of the National Academy of Sciences U.S.A. 113 (12), 1738-1746 (2016).</li><li>M&#252;ller, A., Brandenburg, S., Turkowski, K., M&#252;ller, S., Vajkoczy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+microglia,+and+not+peripheral+macrophages,+are+the+main+source+of+brain+tumor+mononuclear+cells.">Resident microglia, and not peripheral macrophages, are the main source of brain tumor mononuclear cells.</a> International Journal of Cancer. 137 (2), 278-288 (2014).</li><li>McHarg, S., Clark, S. J., Day, A. J., Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+macular+degeneration+and+the+role+of+the+complement+system.">Age-related macular degeneration and the role of the complement system.</a> Molecular Immunology. 67 (1), 43-50 (2015).</li><li>Nozaki, 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=Drusen+complement+components+C3a+and+C5a+promote+choroidal+neovascularization.">Drusen complement components C3a and C5a promote choroidal neovascularization.</a> Proceedings of the National Academy of Sciences U.S.A. 103 (7), 2328-2333 (2006).</li><li>Tsutsumi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+critical+role+of+ocular-infiltrating+macrophages+in+the+development+of+choroidal+neovascularization.">The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization.</a> Journal of Leukocyte Biology. 74 (1), 25-32 (2003).</li><li>Subhi, 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=Association+of+CD11b++Monocytes+and+Anti&#8211;Vascular+Endothelial+Growth+Factor+Injections+in+Treatment+of+Neovascular+Age-Related+Macular+Degeneration+and+Polypoidal+Choroidal+Vasculopathy.">Association of CD11b +Monocytes and Anti&#8211;Vascular Endothelial Growth Factor Injections in Treatment of Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy.</a> JAMA Ophthalmology. 137 (5), 515-518 (2019).</li><li>Singh, 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=Altered+Expression+of+CD46+and+CD59+on+Leukocytes+in+Neovascular+Age-Related+Macular+Degeneration.">Altered Expression of CD46 and CD59 on Leukocytes in Neovascular Age-Related Macular Degeneration.</a> AJOPHT. 154 (1), 193-199 (2012).</li><li>Kwak, N., Okamoto, N., Wood, J. M., Campochiaro, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+is+major+stimulator+in+model+of+choroidal+neovascularization.">VEGF is major stimulator in model of choroidal neovascularization.</a> Investigative Ophthalmology &amp; Visual Science. 41 (10), 3158-3164 (2000).</li><li>Shah, R. S., Soetikno, B. T., Lajko, M., Fawzi, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Mouse+Model+for+Laser-induced+Choroidal+Neovascularization.">A Mouse Model for Laser-induced Choroidal Neovascularization.</a> Journal of Visualized Experiments. (106), e53502 (2015).</li><li>Mahajan, V. B., Skeie, J. M., Assefnia, A. H., Mahajan, M., Tsang, 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=Mouse+Eye+Enucleation+for+Remote+High-throughput+Phenotyping.">Mouse Eye Enucleation for Remote High-throughput Phenotyping.</a> Journal of Visualized Experiments. (57), e3814 (2011).</li><li>Makinde, H. 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=A+Novel+Microglia-Specific+Transcriptional+Signature+Correlates+with+Behavioral+Deficits+in+Neuropsychiatric+Lupus.">A Novel Microglia-Specific Transcriptional Signature Correlates with Behavioral Deficits in Neuropsychiatric Lupus.</a> Frontiers in Immunology. 11, 338-419 (2020).</li><li>Sakurai, E., Anand, A., Ambati, B. K., van Rooijen, N., Ambati, J. <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+inhibits+experimental+choroidal+neovascularization.">Macrophage depletion inhibits experimental choroidal neovascularization.</a> Investigative Ophthalmology &amp; Visual Science. 44 (8), 3578-3585 (2003).</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 (1), 14-20 (2014).</li><li>Liyanage, S. 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=Flow+cytometric+analysis+of+inflammatory+and+resident+myeloid+populations+in+mouse+ocular+inflammatory+models.">Flow cytometric analysis of inflammatory and resident myeloid populations in mouse ocular inflammatory models.</a> Experimental Eye Research. 151, 160-170 (2016).</li><li>Schmutz, S., Valente, M., Cumano, A., Novault, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Cell+Suspensions+Isolated+from+Solid+Tissues+by+Spectral+Flow+Cytometry.">Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry.</a> Journal of Visualized Experiments. (123), e55578 (2017).</li></ol>

多参数流细胞学允许对复杂组织中的多个细胞类型进行定量分析。在本报告中，我们描述了在鼠眼激光损伤后，单核噬细胞群（包括单核细胞、树突细胞和巨噬细胞子集）的流动细胞测定。Liyanage等人最近报告了一种类似的盖特策略，以检测嗜中性粒细胞、嗜酸杆菌、淋巴细胞、DCs、巨噬细胞、渗透巨噬细胞和单核细胞27。我们的量子策略主要因添加 CD64 而有所不同。Liyanage等人?...

与生俱来的免疫系统包括多种细胞类型，刺激补充激活和炎症。与生俱来的免疫细胞包括天然杀手（NK）细胞、乳腺细胞、嗜血杆菌、嗜酸杆菌、嗜中性粒细胞和单核噬细胞。单核噬细胞由单细胞、巨噬细胞和支气管细胞组成，与多种眼科疾病（包括尿毒症、糖尿病视网膜病变和年龄相关的黄斑变性（AMD）1）的病理生理学有关。在本协议中，我们将在血管AMD2小鼠模型中采用多参数流细胞学分析，重点识别单核噬细胞。此协议适合糖尿病视网膜病变和/或尿道炎的鼠标模型，但由于这些疾病的系统性，建议进行更广泛的眼科解剖。
单核噬细胞表示重叠的细胞表面标记。长效组织驻留巨噬细胞和微胶质源自蛋黄囊衍生红细胞原体3，同时回收巨噬细胞和树突细胞从骨髓衍生的大噬细胞树突4。单细胞、巨噬细胞和突触细胞常见的小鼠细胞表面标记包括CD45、CD11b 5、F4/806、Cx3cr17和细胞内标记Iba18。为了克服这一挑战，对来自多个组织的巨噬细胞、单核细胞和突触细胞的转录分析将CD64定义为大噬细胞特异性细胞表面标记6。巨噬细胞在虹膜，胆囊，胆汁身体和视神经在健康的眼睛3描述。或者，突起细胞识别更加困难：最具体的突起细胞识别方法需要使用Zbtb46-GFP记者鼠标9进行命运映射。独立于此记者行，表达CD11c和MHCII结合缺乏CD64可以识别潜在的突起细胞6，10。在正常眼睛11的角膜、结膜、虹膜和胆囊中已经发现了腺细胞。微胶质是位于视网膜的专门巨噬细胞，受视网膜屏障保护，并来自蛋黄囊祖细胞12。因此，视网膜微胶质可以通过CD45表达13的昏暗水平和Tmem119的高水平，作为流细胞抗体14，从单细胞衍生的巨噬细胞中分化出来。然而，在微胶质活化后，CD45可以向上调节15，Tmem119可以被下调节3，这显示了微胶质生物学的复杂性，这可能与AMD及其小鼠模型有关。最后，单核细胞可以分为至少两个亚型，包括古典和非经典。经典单核显示CCR2+Ly6C高CX3CR1低表达，非经典单核显示CCR2-Ly6C低CX3CR1高标记5。
由于对标记表达进行定量分析的必要性，即高与低/低水平，多参数流细胞学是单细胞、巨噬细胞、微胶质和眼睛和其他组织树突细胞之间区别的理想方法。其他优势包括识别亚种群、使用荧光激活细胞分拣 （FACS） 对细胞群进行转录或蛋白分析的能力以及命运映射。多参数流细胞学的主要缺点是缺乏组织结构。这可以通过眼科解剖到各种眼科分组来克服：角膜、结膜、虹膜、镜片、视网膜和胆囊硬化复合体。此外，可以进行确认性免疫荧光成像，但受标记数量和缺乏强健量定量的限制。
基因组广泛关联研究已经将多个补充基因与AMD16联系起来。补充活化导致过敏性拉托辛生产，白细胞招募，并由此产生的炎症。在补充受体缺陷小鼠，激光损伤减少单核噬细胞招募和激光诱导胆囊血管化（CNV）区域17。同样，C-C主题化学素受体2（CCR2）淘汰小鼠，这是缺乏单核细胞招募组织，表明减少单核噬细胞招募和激光诱导的CNV区域18。这些数据链路补充和单核噬细胞与实验CNV和可能的血管AMD。为了支持这种联系，补充受体在血管AMD19，20患者的外周血液单细胞上受到监管。这些数据表明AMD和单核噬细胞之间有很强的联系。
在这份手稿中，我们将利用实验激光诱导CNV模型，利用多参数流细胞学来描述小鼠眼中的单核噬细胞群。激光诱导的CNV是神经血管AMD的标准小鼠模型，它证明了目前一线神经血管AMD治疗21的疗效。此协议将描述小鼠眼睛的电子核化、眼部解剖、消化成单细胞悬浮、抗体染色、使用单色控制确定激光电压以及使用荧光减去一 （FMO） 控制的门禁策略。有关激光诱导的 CNV 模型的详细信息，请参阅之前的出版物22。使用此协议，我们将定义微胶质、单核细胞、突起细胞和巨噬细胞群。此外，我们将使用 MHCII 和 CD11c 进一步定义激光诱导 CNV 模型中的宏噬细胞子集。

<table>
	<tbody>
		<tr>
			<td>0.6 ml microcentrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>05-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml microcentrifuge tubes</td>
			<td>Costar</td>
			<td>3620</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml pipettes</td>
			<td>Fisher Scientific</td>
			<td>431031</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conicals</td>
			<td>ThermoFisher</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>1x HBSS, 500 ml</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5 ml syringe</td>
			<td>Henke Sass Wolf</td>
			<td>7886-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20% paraformaldehyde solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713-S</td>
			<td></td>
		</tr>
		<tr>
			<td>25 ml pipettes</td>
			<td>Fisher Scientific</td>
			<td>431032</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G needle</td>
			<td>Exel</td>
			<td>26437</td>
			<td></td>
		</tr>
		<tr>
			<td>3ml luer lock syringe</td>
			<td>Fisher Scientific</td>
			<td>14955457</td>
			<td></td>
		</tr>
		<tr>
			<td>41 x 41 x 8 mm polystyrene weigh dish</td>
			<td>Fisher Scientific</td>
			<td>08-732-112</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conicals</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well, U-bottom assay plate without lid</td>
			<td>Falcon</td>
			<td>353910</td>
			<td></td>
		</tr>
		<tr>
			<td>Amine reactive beads</td>
			<td>ThermoFisher</td>
			<td>A10628</td>
			<td></td>
		</tr>
		<tr>
			<td>Analysis software</td>
			<td>FlowJo</td>
			<td>v 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rat and anti-hamster Ig kappa bead set</td>
			<td>BD Biosciences</td>
			<td>552845</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice</td>
			<td>Jackson Laboratory</td>
			<td>664</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>Invitrogen</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>Compensation beads</td>
			<td>ThermoFisher</td>
			<td>01-1111-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Count beads</td>
			<td>ThermoFisher</td>
			<td>01-1234-42</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>Fisher Scientific</td>
			<td>16-100-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Digestion enzyme</td>
			<td>Sigma Aldrich</td>
			<td>5401020001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>National Optical and Scientific Instruments, Inc.</td>
			<td>DC3-420T</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociation tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-095-937</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Diva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fc block, rat anti-mouse CD16/CD32</td>
			<td>BD Biosciences</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Integra Miltex</td>
			<td>17035X</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow buffer</td>
			<td>Miltenyi Biotec</td>
			<td>130-091-221</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow tubes</td>
			<td>Falcon</td>
			<td>352008</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamster anti-mouse CD11c BV421</td>
			<td>BD Biosciences</td>
			<td>562782</td>
			<td></td>
		</tr>
		<tr>
			<td>Ice bucket</td>
			<td>Fisher Scientific</td>
			<td>07-210-123</td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead dye</td>
			<td>ThermoFisher</td>
			<td>65-0866-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysing solution, 10x solution</td>
			<td>BD Biosciences</td>
			<td>555899</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro titer tube and rack</td>
			<td>Fisher Scientific</td>
			<td>02-681-380</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse CD64 PE</td>
			<td>BioLegend</td>
			<td>139304</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-mouse NK1.1 PECF594</td>
			<td>BD Biosciences</td>
			<td>562864</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipet tips</td>
			<td>Denville Scientific</td>
			<td>P2404</td>
			<td></td>
		</tr>
		<tr>
			<td>P1000 pipettor</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipet tips</td>
			<td>eppendorf</td>
			<td>22491806</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipettor</td>
			<td>Gilson</td>
			<td>FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>P20 pipettor</td>
			<td>Gilson</td>
			<td>FA10003M</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipet tips</td>
			<td>Denville Scientific</td>
			<td>P2401</td>
			<td></td>
		</tr>
		<tr>
			<td>P200 pipettor</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet man</td>
			<td>Fisher Scientific</td>
			<td>FB14955202</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse B220 PECF594</td>
			<td>BD Biosciences</td>
			<td>562313</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD11b APC-Cy7</td>
			<td>BD Biosciences</td>
			<td>557657</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD19 AlexaFluor 700</td>
			<td>BD Biosciences</td>
			<td>557958</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD19 PE</td>
			<td>BD Biosciences</td>
			<td>553786</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD4 PECF594</td>
			<td>BD Biosciences</td>
			<td>562314</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45 FITC</td>
			<td>ThermoFisher</td>
			<td>11-0451-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD45 PE-Cy7</td>
			<td>BD Biosciences</td>
			<td>552848</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse CD8 PECF594</td>
			<td>BD Biosciences</td>
			<td>562315</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly6C</td>
			<td>BD Biosciences</td>
			<td>561085</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Ly6G PECF594</td>
			<td>BD Biosciences</td>
			<td>562700</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse MHC II AlexaFluor 700</td>
			<td>BioLegend</td>
			<td>107622</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Siglec F PECF594</td>
			<td>BD Biosciences</td>
			<td>562757</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat anti-mouse Tim4 AlexaFluor647</td>
			<td>BD Biosciences</td>
			<td>564178</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Labnet</td>
			<td>311DS</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>15024-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cell strainer, 40 mm nylon mesh</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile water, 500 ml</td>
			<td>Gibco</td>
			<td>A12873-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging bucket centrifuge</td>
			<td>eppendorf</td>
			<td>5910 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Gibco</td>
			<td>15250061</td>
			<td></td>
		</tr>
	</tbody>
</table>

digestion of whole mouse eyes for multi-parameter flow cytometric analysis of mononuclear phagocytes

与生俱来的免疫系统在眼部病理生理学中起着重要作用，包括尿道炎、糖尿病视网膜病变和与年龄相关的黄斑变性。与生俱来的免疫细胞，特别是单核噬细胞，表达重叠的细胞表面标记，这使得识别这些群体成为一项挑战。多参数流细胞学允许同时对多个细胞表面标记进行定量分析，以区分小鼠眼睛中的单细胞、巨噬细胞、微胶质和树突状细胞。此协议描述了整个小鼠眼睛的电子核化、眼部解剖、消化成单细胞悬浮物以及骨髓细胞标记的单细胞悬浮物的染色。此外，我们解释了使用单色控制确定电压的正确方法，以及使用荧光减去一个控制来描述正门的正确方法。多参数流细胞学的主要局限性是缺乏组织结构。这种限制可以通过单个眼室的多参数流动细胞学或免费免疫荧光染色来克服。然而，免疫荧光因缺乏定量分析和大多数显微镜上的荧光素数量减少而受到限制。我们描述了在激光诱导的胆囊化中使用多参数流细胞学来提供单核噬细胞的高度定量分析。此外，多参数流细胞学可用于识别巨噬细胞子集、命运映射和细胞分拣，用于转录学或前列腺学研究。

图 1显示了 SCC 和红色激光细胞的无补偿频率直方图：Alexa647、Alexa700 和 APC-Cy7。在图 1A中，洋红色线描绘了 Alexa647 的 SCC 峰值，而青色线显示了预期的 0.5 日志差异。请注意，Alexa700 和 APC-Cy7 的峰值位于青色线的左侧（不太亮）。还要注意的是，这些细胞都在洋红色线的左边。SCC 峰值右侧的任何单元格均未得到补偿。据了解，根据荧色染料的化学成分?...

Watch this Scientific Journal Video about Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes at JoVE.com

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

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

该方案提供了一种将整个眼睛消化成单细胞悬浮物的方法，用于多参数流动细胞学分析，以确定特定的眼单核噬细胞群，包括单细胞、微胶质、巨噬细胞和树突细胞。

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

Research

JoVE Journal

Immunology and Infection

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

主要鼠Ⅱ型肺泡上皮细胞的功能及分子生物学研究的流式细胞仪分离

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

科研

免疫与感染

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

对于纳米颗粒疫苗接种后的分析抗原特异性CD8 + T细胞应答整体动物成像和流式细胞技术

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

分离和流式细胞仪分析从缺血小鼠脑免疫细胞

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

分离和胶质瘤浸润外周血单个核细胞的流式细胞仪分析

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

一个简单的流式细胞仪法测量葡萄糖的摄取和葡萄糖转运表达单核细胞亚群的全血

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

粒子结合Bet v 1的变应原PM10的流式细胞分析

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

人全血中NK细胞杀伤活性的流式细胞仪分析

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

人类宫颈的γδT细胞的分离和流式细胞仪分析

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...