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。 注:可进行心肌输液，以去除不在眼组织中的循环白细胞。在这个实验中，全身注水不会减少在未经治疗或激光治疗的整个眼睛中检测到的微胶质、单细胞、巨噬细胞或神化细胞的数量。因此，这些细胞类型大多位于眼科组织中，此步骤是可选的。</li>
	<li>要使眼睛产生核核，在眼睛插座附近向下推，同时在眼睛从插座中伸出时轻轻地将一对弯曲的钳子放在眼睛下面。捏钳子关闭眼睛下没有挤压实际的眼睛。从周围的结膜（请参阅之前的出版物）23。</li>
	<li>横向拉眼，直到视神经是分离眼睛的唯一剩余连接。视神经经常与紧张分离，但一个小剪刀有时是必要的，以削减视神经。将眼睛放在冷1x汉克的缓冲盐水溶液（HBSS）与钙和镁在1.7 mL微中心管。</li>
</ol>2. 眼组织消化
<ol><li>在寒冷的HBS中准备含有0.1毫克/mL消化酶和0.2毫克/mL DNase的消化缓冲液。最初在2mL的无菌水中重组5毫克消化酶。在 HBS 中准备 10 毫克/mL DNase 的初始稀释。每10 mL的消化缓冲（足够3只动物）混合9.4 mL的冷HBSS与0.4 mL的重组消化酶和0.2毫秒的稀释DNase I。 注:这些浓度通过多个实验经验确定，以提供单细胞抗体染色的最佳水平，同时减少细胞死亡。拼贴酶D被尝试作为消化酶的替代品，降低细胞存活率和减少CD45+ 细胞。有关优化消化的迭代过程的更多详细信息，请参阅 讨论 。</li>
	<li>在总放大倍数为 10 倍的解剖显微镜下，使用冷 HBSS 中的细钳和弹簧剪刀去除剩余的结膜和视神经。</li>
	<li>将干净的眼睛移到干燥的解剖盘或小型称重船上。通过装有30G针的注射器将消化缓冲液的0.15-0.20 mL/眼睛注射到视腔中，然后通过视觉轴在眼睛中穿孔两处伤口，距离第一个伤口90°以消化缓冲器出口。</li>
	<li>使用弹簧剪刀和细钳子，所有小于 0.5 mm x 0.5 mm 的片断，将整只眼睛切碎。通过钳子将透镜组织与钝性机械干扰分离，以防止在随后的步骤中堵塞移液器尖端。对于每个样品，用0.75 mL的消化缓冲液将钳子和剪刀分别冲洗成小菜。每个样品使用新菜。</li>
	<li>使用 P1000 移液器将盘中的内容转移到冰上的分离管中，注意在移液器传输中不会丢失任何材料。用额外的 1.5 mL 消化缓冲液冲洗菜，使分离管的总体积达到 3.25-3.50 mL。</li>
	<li>将反向分离管放置在电子分离器上并运行周期"m_brain_03_01"，在室温下在 200 rpm 下由 1 分钟的单向旋转组成，这是一个预加载程序。确保所有组织都位于分离管的底部，与消化缓冲器和转子接触，用于此步骤和所有剩余步骤。</li>
	<li>在 37 °C 的 200 rpm 时，将分离管放置在摇动孵化器中 30 分钟。</li>
	<li>重复步骤 2.6 和 2.7 一个额外的时间。在第二次30分钟的孵化后，使用电子分离器进行最后一次机械消化，就像第2.6步一样。</li>
	<li>通过添加 10 mL 的冷流缓冲器并在冰上放置管子来停止反应。</li>
</ol>3. 准备细胞悬架
<ol><li>要创建单细胞悬架，通过放置在 50 mL 圆锥形离心管顶部的 40μm 过滤器倒入停止的眼睛消化反应。使用 2.5 mL 注射器中的柱塞，将剩余未消化的眼组织通过过滤器推入。</li>
	<li>使用 10 mL 的流缓冲器和冲洗过滤器清洗分离管，然后使用相同的柱塞再次通过过滤器传递未消化的眼组织件。</li>
	<li>使用5 mL的流缓冲区重复步骤3.2。</li>
	<li>用最终的 10 mL 流缓冲器清洗分离管和过滤器。 注:每个样本的消化和流量缓冲液总体积约为38-40 mL。</li>
	<li>在4°C下将锥形管在400 x g 上离心10分钟。在不干扰细胞颗粒的情况下去除超纳坦。</li>
	<li>通过在无菌水中加入 10 倍裂解溶液，准备 1x 裂解液。通过将管子轻拂到另一根管子上来分解颗粒。要溶解红血球，请添加 1 mL 的 1x 裂解溶液，用于 30 s，在室温下旋转 （RT）。</li>
	<li>用20 mL的流缓冲区停止反应。</li>
	<li>在4°C下将锥形管在400 x g 上离心10分钟。在不干扰细胞颗粒的情况下去除超纳坦。</li>
	<li>通过将管子轻拂到另一根管子上来分离颗粒。每管添加5 mL的冷1x HBSS。</li>
	<li>离心管在400 x g 10分钟，在4°C。有抱负的超级纳坦。</li>
</ol>4. 单核噬细胞细胞悬浮物的污渍
<ol><li>通过稀释冷 HBS 中的活/死染料 1:5，000，每个样品准备 0.5 mL 的活/死污渍。</li>
	<li>使用 P1000 移液器将活/死污渍添加到每个样品中，确保颗粒完全分离。将样品转移到 1.2 mL 微滴度管中。</li>
	<li>使用血细胞计或自动细胞计数器（见4.4）来计算细胞。在黑暗的室温下用活/死污渍孵化样品15分钟。</li>
	<li>使用 Trypan 蓝色计数细胞以确定每个样本的活细胞数。 注:通常情况下，10-12周大的C57BL/6J小鼠的两只眼睛含有少于2 x 106 个活细胞。</li>
	<li>通过添加 400μL 的冷流缓冲器来清洗样品。微型滴度管架中的离心管在 400 x g 下，在 4 °C 下 10 分钟。在不干扰细胞颗粒的情况下吸食超纳坦。</li>
	<li>完全在 500μL 的冷流缓冲区中重新悬念细胞。微型滴度管架中的离心管在 400 x g 下，在 4 °C 下 10 分钟。在不干扰细胞颗粒的情况下吸食超纳坦。</li>
	<li>在 Fc块的 50 μL 中阻挡多达 5 x 106个活细胞（纯化大鼠反鼠 CD16/CD32 冷流缓冲区中 1:50 稀释）。如果活细胞超过 5 x 106，则适当增加体积。</li>
	<li>将 Fc 块添加到颗粒中，确保样品完全分离。在4°C下孵化20分钟。</li>
	<li>添加 50 μL 的抗体染色溶液（参见 表 1， 了解溶液中包含的每个抗体的选择和数量），每 5 x 106 活细胞直接添加到 Fc 块中的细胞中并完全混合。在4°C下孵化30分钟。 注:抗体数量取决于流量计配置，每个实验室和仪器应独立进行滴定。有关过滤器和后视镜的仪器配置，请参阅 表 2。 要对抗体进行滴定，从制造商的建议开始，并计算染色指数。执行不同抗体浓度的测试（高于和低于制造商的建议），并选择保持染色指数的最低抗体浓度。此外，确保比单色控制更不亮的正染色。使用未染色的细胞，以确保负染色细胞是规模和有峰值在或小于1 x 102。使用荧光减去一个控制来定义正染色细胞。</li>
	<li>通过添加 700μL 的冷流缓冲器来清洗样品。微型滴度管架中的离心管在 400 x g 下，在 4 °C 下 10 分钟。在不干扰细胞颗粒的情况下吸食超纳坦。</li>
	<li>可选地，抗体染色后，样品可以在流缓冲区用500μL的2%副醛固定。在室温下将样品固定15分钟。然后，执行 4.10 中描述的一次洗涤。将固定样品存放在4°C。应在流细胞学数据收集当天添加数珠。固定样品应在 1-2 天内在流式细胞计上运行。 警告:甲醛是一种腐蚀性和健康危害。</li>
	<li>使用 500μL 的冷流缓冲器再次清洗样品。微型滴度管架中的离心管在 400 x g 下，在 4 °C 下 10 分钟。吸食一半的超生剂，而不会干扰细胞颗粒。</li>
	<li>重新悬念颗粒并转移到 96 井 U 底测定板。离心板在400 x g 5分钟在4°C。要倒掉超沉剂，将板翻过来，在一次强烈的摇晃中将液体分离成沉盆，而不会干扰颗粒。</li>
	<li>在一个新的1.2 mL微型滴度管中，放置50μL的涡流计数珠。</li>
	<li>在 200μL 冷流缓冲区 96 个井板中重新悬索颗粒。从上一步在珠子顶部添加细胞混合物。在总体积为250μL/微滴度管的流量计上运行。</li>
</ol>5. 流细胞学数据的收集
<ol><li>打开并准备流量计。此处列出的说明用于修改后的四个激光测速仪（表2）。</li>
	<li>运行一个测试样本，其中包括每个样本中的一个小方位，以确保每个检测器的所有细胞都具有规模。调整电压，使所有细胞都达到规模。</li>
	<li>对染色鸡尾酒中使用的每种荧光素（表3）运行单色控制（SCC）。将微型滴度管放入更大的 5 mL 聚苯乙烯管中，放置在流细胞仪阶段。 注:根荧光剂，如BV421（太平洋蓝）、FITC、PE、Alexa647（APC）和Alexa700不需要与鸡尾酒相同的抗体（即CD19 PE用于CD64 PE）。但是，由于合并荧光素的潜在分离，PE-CF594、PE-Cy7 或 APC-Cy7 等串联荧光剂对 SCC 的抗体要求与鸡尾酒相同。<ol>
<li>为 BV421 创建 SCC，在 1.2 mL 滴度管中添加 2 滴补偿珠，以及 1 μL 的仓鼠防鼠 CD11c BV421。补偿珠的使用是因为CD11c BV421是IgG兰姆达亚型，而不是5.3.3中描述的卡帕。在4°C下孵化15分钟。添加0.2 mL的流量缓冲区。</li>
		<li>通过从胺活性珠子中添加一滴正染色珠（绿色帽），然后添加 1μL 的活/死染料，创建活/死染料的 SCC。在黑暗的室温下孵化15分钟。从胺活性珠子中加入一滴负珠（白帽）。添加0.2 mL的流量缓冲区。</li>
		<li>通过从抗鼠和抗仓鼠 Ig kappa 珠集中添加一滴正染色珠（绿色帽），创建剩余的 SCC，然后在 1.2 mL 滴度管中列出抗体数量（表 3）。在4°C下孵化15分钟。从反鼠和反仓鼠 Ig kappa 珠集中添加一滴负珠（白帽）。添加0.2 mL的流量缓冲区。</li>
		<li>漩涡所有珠子混合物完全。在4°C存储SC长达3天。 注:荧光记者小鼠的SCC是未染色的细胞。</li>
		<li>运行SC时，设置电压，使SCC样品的峰值大于同一检测通道中细胞样本的任何荧光。此外，应设置电压，以便任何 SCC 在兴趣通道中的直方图峰值与任何其他通道（图 1）之间至少有 0.5日志差。</li>
	</ol>
</li>
	<li>运行"低"保持事件/秒低于4000的样本。如果流量不能适当降低，则用额外的冷流缓冲器稀释样品。记录添加的音量，因为计数计算是必要的（见代表结果）。</li>
	<li>每个样本至少收集 3 x 106 个事件。这可能高于细胞数，因为颜料颗粒和碎屑计数为细胞计上的事件。</li>
	<li>在第 6.4 节中，记录除 Live/Dead 以外的每个荧光减去一 （FMO）， 以获得适当的门禁策略。FMO 是除一种外已染色所有荧光素的样本。</li>
	<li>根据制造商或设施说明清洁和关闭流量计。</li>
</ol>6. 流细胞学数据注释
<ol><li>使用分析软件打开新的工作表。从细胞计导入FCS文件，用于所有样品和单色控制。</li>
	<li>使用SCC创建补偿矩阵。</li>
	<li>将补偿矩阵应用于您的染色样品和 FMO。</li>
	<li>使用 FMO 确定正染色以绘制门。</li>
</ol>

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作者没有什么可透露的。

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等人将DC识别为CD4-CD8-B220-CD11b+CD11c+细胞，将巨噬细胞识别为CD4-CD8-B220-CD11b-CD11c-NK1.1-Ly6G细胞。我们确定DC为CD4-CD8-B220-Ly6G-SiglecF-NK1.1-CD11b+CD11c+CD64-细胞和巨噬细胞为CD4-CD8-B220-Ly6G-西格勒克F-NK1.1-CD11b+CD64+细胞。使用CD64区分直流与巨噬细胞是既定的6，这种策略使我们能够识别巨噬细胞异质性，包括CD11c+巨噬细胞。此方法的主要局限性是，它不提供有关组织结构或眼睛内特定细胞位置的数据。为了确定这些细胞是在视网膜，胆囊，虹膜，或其他眼结构，我们的协议可以结合组织学方法或修改，包括更广泛的眼科解剖，以单独运行多参数流细胞在每个眼科子腔。
我们优化了整个小鼠眼睛消化方案，以防止解剖产生的任何差异。例如，切除角膜、虹膜和镜片的后眼杯可能会从保留的虹膜或角膜材料（解剖不足）中产生变异性，或增加激光对正常后眼杯区域（过度解剖）的伤害。我们比较控制无玻璃和激光眼睛检测单核噬细胞的渗透，因为视网膜损伤。对于系统性疾病对眼睛的影响（即尿炎和糖尿病）的分析，单个眼科分组分析是必不可少的。在协议第2节中，消化方法是最关键的，而第3-5节的其他步骤已成功地用于多个组织类型24。我们通过一种迭期方法得出了消化条件:（1）化学消化方法（拼贴酶D与消化酶）的测试），（2）化学消化长度的测定（30、60和90分钟），以及（3）增加机械消化（无、一次、两次、三次）。在每一步，我们判断消化条件的成功，由细胞生存能力和CD45HiCD64+（渗透巨噬细胞）细胞的数量。我们发现消化酶（见材料补充表）比拼贴酶D回收更多的活细胞，并尝试一半的消化酶浓度，导致渗透巨噬细胞更少。接下来，60分钟的化学消化与机械消化几乎加倍的渗透巨噬细胞种群。三个机械消化是最佳的25-30%的活细胞的单体和最渗透的巨噬细胞种群。最后，更快的机械消化条件导致活细胞和巨噬细胞的严重损失。
将来，我们建议通过去除角膜、虹膜和镜片来扩展这种方法，然后分别消化视网膜和后眼杯（硬膜、胆囊和视网膜色素上皮）。通过这种修改，我们预计会减少消化条件，因为镜片和角膜的蛋白质和密集特性分别。这些减少可能包括减少消化酶浓度，切换到拼贴酶，减少消化时间，或减少机械消化量。我们预计后眼杯的消化条件会全面降低，视网膜本身的消化条件将进一步降低。
其他未来方向包括将我们传统的多参数流细胞学面板扩展到传统的 6 激光仪器或光谱流细胞学。光谱流细胞学允许在整个光谱中检测荧光，而不是由可用的过滤器决定。光谱流细胞学导致更具体的颜色检测。然而，光谱流细胞学需要遵循一套全新的考虑，超出了本手稿的范围。欲了解更多详情，请参阅最近 JoVE 第28条。这6台激光仪器包括一个紫外激光器，它增加了6-9个探测器。在向鼠标眼流细胞学面板添加标记时，我们建议检测更多眼细胞类型。例如，可以分离系门，独立检测嗜中性粒细胞、嗜酸杆菌、乳腺细胞、NK细胞和淋巴细胞。建议增加单核噬细胞的子集检测，因为处于稳定状态的巨噬细胞数量很少。在添加新的抗体时，我们建议小心抗体滴定。
总之，我们提出了一种适应性强的方法，用于检测小鼠眼中的单核噬细胞群。由于单细胞、树突细胞、巨噬细胞和微胶质的细胞表面标记高度重叠，多参数流细胞学是检测这些细胞群的最佳方法。流细胞学可用于细胞类型分析、命运映射和/或细胞分拣，用于转录或蛋白皮评估。它的主要局限性是缺乏组织结构，关键发现可以用传统的组织学来证实。

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，特定通道会溢出到其他通道（即 紫罗兰激光:BV421-+V500，黄绿色激光:PE->PE-CF594，红色激光:亚历克萨647-亚历克萨700，亚历克萨700-=APC-Cy7，交叉激光:PE-Cy7-+APC-Cy7）。如果上述任何一个条件均未满足，则一个通道的电压可以向上调整，而任何溢油通道都可以向下移动。有关红色激光的其他示例，请参阅图 1B 和图 1C。 一旦确定并记录电压，所有样品都必须使用电压参数运行。设置向导的补偿存在，并且可能有帮助。
数珠识别对于细胞计数的量化是必要的。图 2A显示了 FSC-A vs SSC-A 属性，用于单个鼠标的两只眼睛分析的所有事件。重要的是要调整SSC电压，以确保您的计数珠是可见的SSC-A高和FSC-A低事件非常紧密的集合。通过绘制 PE 与 APC-Cy7（图 2B），对珠子计数进行了清理和确认。清洁珠子是一个紧密的PE+和APC-Cy7-事件集群。
接下来，从所有事件中都发现了单体细胞和活细胞。单打是通过绘制 Fsc - h vs Fsc - a 来确定的。单体在这两种特性中呈正相关关系，而双胞胎和三胞胎的FSC-A比FSC-H（图2C）大。通过绘制活/死与FSC-A绘制活细胞。死细胞是活的/死的阳性，细胞表现出比碎片更大的FSC-A（图2D）。请注意，计数珠是活/死+， 并在此步骤中被删除。
图 3显示了从活的单核噬细胞中划定单核噬细胞的初始量子策略。现场单打使用CD45与CD11b情节（图3，左）可视化。请注意，选择 CD45+CD11b- （主要是 B 细胞和 T 细胞）、CD45昏暗CD11b+ （假定微胶质） 和 CD45+CD11b+细胞 （渗透免疫细胞，用激光 [图 3B， 左] 增加）。CD45+细胞在CD45 FMO中不存在，这证实了门的选择（图3C，左图）。接下来，嗜中性粒细胞， 通过绘制CD45+使用血统门的活体单体（林:Ly6G[嗜中性粒细胞]、B220[B细胞]、NK1.1[NK细胞]、CD4[T细胞]和CD8[T细胞]）与CD11b，排除了嗜酸性粒细胞、B细胞、B细胞、NK细胞和CD8[T细胞]。很容易观察到无激光（图3A，中间）和激光（图3B，中间）组之间的CD11b+Lin细胞的增加。请注意，CD11b+CD11b FMO 中的林细胞（图 3C，中间）中缺乏 CD11b + 林细胞，以及林 FMO 中缺少林+细胞（图 3C，右图）。微胶质可以通过CD45表达13、24的数量从单核噬细胞中分离出来。CD11b+Lin-使用 CD11b 与 CD45 图进行可视化，以识别 CD45昏暗假定微胶质和 CD45高渗透单核噬细胞。CD45高单核噬细胞的相对增加在激光处理小鼠（图3A，B，右）中是明显的。
图4识别出微胶质、三个巨噬细胞子集、单核细胞和支离细胞。微胶质是通过在CD64与MHCII图（图4，左面板）上绘制CD45暗细胞来确定的。微胶质已被证明是CD64+MHCII低之前13。Microglia与激光相对没有变化，在CD64 FMO中不存在（图4A，C左）。CD45高细胞下一步绘制在同一 CD64 与 MHCII 图形上。CD64+MHCII-巨噬细胞 （MHCII- Mac），CD64-MHCII-单核细胞和 MHCII+细胞被识别（图 4A，B，左中）。请注意MHCII+细胞在MHCII FMO中不存在（图4C，左中）。MHCII +细胞被进一步歧视使用 CD64 和 CD11c. CD64+CD11c-巨噬细胞 （CD11c- Mac），CD64+CD11c+大噬细胞 （图 4A， B- 中间偏右） 。请注意CD11c+CD11c+细胞在CD11c FMO中缺失（图4C，右中）。单核细胞被子型为经典Ly6C+或非经典Ly6C- 单核细胞（图4，右）。请注意，Ly6C+单核细胞在Ly6C FMO中不存在（图4C，右图）。
使用以下方程计算每只小鼠（或每两只眼睛）的细胞数:
<img alt="Equation 1" src="/files/ftp_upload/61348/61348equ01v2.jpg">
使用此方法中的卷，方程是:
<img alt="Equation 2" src="/files/ftp_upload/61348/61348equ02.jpg">
细胞计数和珠数将特定于每个样本。珠子的浓度是特定于每个批次的数珠，由制造商在每个小瓶上提供。
巨噬细胞在激光损伤25后渗入胆囊，巨噬细胞耗竭减少CNV区域18。这些较旧的研究依赖于免疫荧光成像，它不能可靠地区分单核噬细胞，或更少的流动细胞学标记物进行巨噬细胞识别。巨噬细胞异质性是天生免疫学中一个新兴的概念，其中巨噬细胞能够根据其起源和组织微环境12、26执行多种功能。在激光损伤后的第3天，我们对未经治疗的和激光治疗的老鼠眼睛进行了多参数流细胞学，以确定单核噬菌体和巨噬细胞异质性。激光治疗分别增加了MHCII-，CD11c-和CD11c+巨噬细胞7.0-25.6，2.8-7.2和3.9-8.2倍（图5A，C）。通过激光（图5F），登德里特细胞计数也上升了4.5-4.7倍。微胶质和单核细胞计数不受激光治疗的影响（图5D，E）。在电子核化之前，无论是否系统注入，均未检测到显著差异。这些结果表明，在激光损伤后，巨噬细胞数量和突起细胞数量增加，微胶质或单核细胞没有变化。此外，这些数据突出了多参数流细胞学如何检测巨噬细胞异质性。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>抗体或缓冲区</td>
			<td>荧光</td>
			<td>克隆</td>
			<td>每个样本的量（ng）</td>
		</tr>
<tr>
<td>鼠抗鼠 Ly6C</td>
			<td>菲特克</td>
			<td>AL-21</td>
			<td>15</td>
		</tr>
<tr>
<td>鼠标反鼠标 CD64</td>
			<td>体育</td>
			<td>X54-5/7.1</td>
			<td>40</td>
		</tr>
<tr>
<td>鼠抗鼠蒂姆4</td>
			<td>亚历克萨弗鲁尔 647</td>
			<td>RMT4-54</td>
			<td>300</td>
		</tr>
<tr>
<td>仓鼠反鼠标 CD11c</td>
			<td>BV 421</td>
			<td>HL3</td>
			<td>300</td>
		</tr>
<tr>
<td>鼠抗鼠 Ly6G</td>
			<td>PE-CF594</td>
			<td>1A8</td>
			<td>10</td>
		</tr>
<tr>
<td>鼠标反鼠标NK1.1</td>
			<td>PE-CF594</td>
			<td>PK136</td>
			<td>10</td>
		</tr>
<tr>
<td>大鼠反鼠西格莱克 F</td>
			<td>PE-CF594</td>
			<td>E50-2440</td>
			<td>20</td>
		</tr>
<tr>
<td>鼠抗鼠 B220</td>
			<td>PE-CF594</td>
			<td>RA3-6B2</td>
			<td>20</td>
		</tr>
<tr>
<td>鼠抗鼠 CD8</td>
			<td>PE-CF594</td>
			<td>53-6.7</td>
			<td>40</td>
		</tr>
<tr>
<td>鼠抗鼠 CD4</td>
			<td>PE-CF594</td>
			<td>RM4-5</td>
			<td>20</td>
		</tr>
<tr>
<td>鼠抗鼠MHC II</td>
			<td>亚历克萨影响 700</td>
			<td>M5/114.15.2</td>
			<td>2.5</td>
		</tr>
<tr>
<td>大鼠反鼠CD11b</td>
			<td>APC-赛7</td>
			<td>M1/70</td>
			<td>2</td>
		</tr>
<tr>
<td>鼠抗鼠 CD45</td>
			<td>PE-赛7</td>
			<td>30-F11</td>
			<td>6</td>
		</tr>
<tr>
<td>MAC缓冲区</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表1:抗体荧光和克隆（见步骤4.9）。 通常，一个样本是一个或两个消化的眼睛。先将流量缓冲器添加到管子中，然后再添加每个抗体。可以进行稀释，以避免管道太小的体积，同时适当地改变总流量缓冲体积。有关产品编号和公司的材料表，请参阅。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>激光</td>
			<td>PMT 插槽</td>
			<td>滤波器</td>
			<td>镜子</td>
			<td>颜色</td>
		</tr>
<tr>
<td rowspan="2">紫罗兰色 （405nm）</td>
			<td>A</td>
			<td>525/50</td>
			<td>505LP</td>
			<td>V500 或美国之安</td>
		</tr>
<tr>
<td>B</td>
			<td>450/50</td>
			<td></td>
			<td>太平洋蓝、eFluor 450、V450 或 BV421</td>
		</tr>
<tr>
<td rowspan="3">蓝色 （488nm/FSC）</td>
			<td>A</td>
			<td>710/50</td>
			<td>685LP</td>
			<td>珀普 - Cy5.5， 佩尔普， PI 或 7aad</td>
		</tr>
<tr>
<td>B</td>
			<td>525/50</td>
			<td>505LP</td>
			<td>FITC、CFSE、GFP 或亚历克萨影响器 488</td>
		</tr>
<tr>
<td>C</td>
			<td>488/10</td>
			<td></td>
			<td>SSC</td>
		</tr>
<tr>
<td rowspan="3">黄绿色 （561nm）</td>
			<td>A</td>
			<td>780/60</td>
			<td>735LP</td>
			<td>PE-赛7</td>
		</tr>
<tr>
<td>B</td>
			<td>610/20</td>
			<td>600LP</td>
			<td>PE-CF594，切里或DsRed2</td>
		</tr>
<tr>
<td>C</td>
			<td>582/15</td>
			<td></td>
			<td>体育</td>
		</tr>
<tr>
<td rowspan="3">红色 （640nm）</td>
			<td>A</td>
			<td>780/60</td>
			<td>735LP</td>
			<td>APC-Cy7、APC-H7或APC-影响780</td>
		</tr>
<tr>
<td>B</td>
			<td>730/45</td>
			<td>690LP</td>
			<td>亚历克萨影响 700</td>
		</tr>
<tr>
<td>C</td>
			<td>670/30</td>
			<td></td>
			<td>APC 或亚历克萨流体 647</td>
		</tr>
</tbody></table>表2:流动细胞仪的配置。 过滤和镜像设置的四激光细胞计和可用的荧光层，可以检测到每个通道。PMT=光复数管，FSC=向前散射，SSC=侧散射。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>抗体</td>
			<td>荧光</td>
			<td>克隆</td>
			<td>每个 SCC 的金额（ng）</td>
		</tr>
<tr>
<td>鼠抗鼠 CD45</td>
			<td>菲特克</td>
			<td>30-F11</td>
			<td>500</td>
		</tr>
<tr>
<td>鼠抗鼠 CD19</td>
			<td>体育</td>
			<td>1D3</td>
			<td>40</td>
		</tr>
<tr>
<td>鼠抗鼠蒂姆4</td>
			<td>亚历克萨弗鲁尔 647</td>
			<td>RMT4-54</td>
			<td>100</td>
		</tr>
<tr>
<td>仓鼠反鼠标 CD11c</td>
			<td>BV421</td>
			<td>HL3</td>
			<td>200</td>
		</tr>
<tr>
<td>大鼠反鼠西格莱克 F</td>
			<td>PE-CF594</td>
			<td>E50-2440</td>
			<td>40</td>
		</tr>
<tr>
<td>鼠抗鼠 CD19</td>
			<td>亚历克萨影响 700</td>
			<td>1D3</td>
			<td>200</td>
		</tr>
<tr>
<td>大鼠反鼠CD11b</td>
			<td>APC-赛7</td>
			<td>M1/70</td>
			<td>200</td>
		</tr>
<tr>
<td>鼠抗鼠 CD45</td>
			<td>PE-赛7</td>
			<td>30-F11</td>
			<td>200</td>
		</tr>
<tr>
<td>活染料/死染料</td>
			<td>e 影响 506</td>
			<td>不适用</td>
			<td>1 微升</td>
		</tr>
</tbody></table>表3:用于单色控制的抗体荧光和克隆。 每个抗体应添加到适当的补偿珠中，如步骤 5.3.1 - 5.3.3 所述。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61348/61348fig01.jpg"> 图1:为红色激光设置代表电压。（A） 亚历克萨647的单色控制（SCCs）。在左侧显示亚历克萨647的SCC。左中间显示亚历克萨647 SCC泄漏到亚历克萨700通道。Alexa647 SCC 的峰值以洋红色显示，0.5 日志目标差以青色显示。请注意，峰值位于左侧（不太亮），而不是青色线。中间偏右显示亚历克萨647 SCC泄漏到APC-Cy7通道。右图说明了 Alexa647 通道中的细胞。请注意，所有细胞的亮度都低于 SCC（洋红色线）。（B） 亚历克萨700的SCC。中间左显示亚历克萨700的SCC。左侧显示亚历克萨700 SCC泄漏到亚历克萨647通道。中右显示亚历克萨 700 Scc 泄漏到 Apc - cy7 频道。Alexa700 SCC 的峰值以洋红色显示，0.5 日志目标差以青色显示。请注意，峰值位于左侧（不太亮），而不是青色线。右图说明了 Alexa700 通道中的细胞。请注意，所有细胞的亮度都低于 SCC（洋红色线）。（C） Apc - Cy7 的 Scc 。左侧和左中侧分别显示 APC-Cy7 SCC 泄漏到 Alexa647 和 Alexa700。请注意，两个山峰都在青线的左侧。中右显示非加太-Cy7 SCC。右图说明了 APC-Cy7 通道中的细胞。请注意，所有细胞的亮度都低于 SCC（洋红色线）。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61348/61348fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61348/61348fig02.jpg"> 图2:对计数珠、单体和活细胞的代表性识别。（A） 轮廓图上所有单元格的侧散射区域 （SSC-A） 与前方散射区域 （FSC-A）。计数珠子由高 SSC-A、低 FSC-A 和非常紧密的均匀珠子聚类识别。（B） PE vs APC-Cy7 地块，用于清洁珠门。A 和 B 之间的箭头表示仅绘制计数珠正事件。清洁计数珠子由高PE和低APC-Cy7荧光所标定。（C） FSC-高 （FSC-H） vs FSC-区域 （FSC-A） 所有细胞的图示单体细胞在正相关宽线。双倍和其他多重显示比 FSC 高度更大的 FSC 区域。（D）活/死vs FSC-A用于单细胞。活细胞被定义为活/死低和FSC-A阳性。百分比表示父母的百分比。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61348/61348fig02large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61348/61348fig03.jpg"> 图3:单核噬菌体的代表性鉴定。左图:CD45 vs CD11b 伪彩色图的活细胞从无激光（A）， 激光（B），和荧光减去一 （FMO） 控制（C）.左图:CD45+细胞在A-B中定义，在FMO中不存在。请注意，在激光（B）组中CD45+CD11b+细胞的增加。中间:血统（林）标记的伪彩色图与CD45+细胞的CD11b。CD11b+Lin-单元格在 A-B 中划界，在 CD11b 的 FMO 中不存在（底部）。右图:CD11b vs CD45图的CD11b+林-细胞。识别CD45变暗和CD45高细胞。请注意激光（B）组CD45高细胞的增加。百分比表示父母的百分比。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61348/61348fig03large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61348/61348fig04.jpg"> 图4:显微胶质、突起细胞、单核细胞和巨噬细胞子集的代表性识别。左图:CD64 vs MHCII 的 CD45暗细胞的伪彩色图将微胶质定义为 CD64+MHCII低。请注意未激光（A）和激光（B）组的微胶质量相似，FMO控制（C）中缺乏微胶质。中左:CD64 vs CD45高细胞的MHCII伪彩色图将MHCII-巨噬细胞定义为CD64+MHCII-，单核细胞定义为CD64-MHCII-和MHCII+细胞。请注意，激光组 （B） 中 MHCII - 巨噬细胞的增加、FMO（C、中间）和 CD64 FMO（C、左）中缺乏 MHCII® 细胞有助于确定 CD64+和 CD64-细胞的截止时间。中间偏右:CD64 vs CD11c MHCII+细胞的伪彩色绘图。CD11c-巨噬细胞被定义为CD64+CD11c-，CD11c+巨噬细胞被定义为CD64+CD11c+，突起细胞（DCs）被定义为CD64-CD11c+。大噬细胞子集和支离细胞都通过激光（B）增加。请注意FMO（C）中缺少CD11c+细胞。右图:Ly6C vs Tim4单核细胞的伪彩色绘图。经典 Ly6C+和非经典 Ly6C-单核细胞被识别。请注意，Ly6C+单核细胞在Ly6C FMO（C）中不存在。百分比表示父母的百分比。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61348/61348fig04large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61348/61348fig05.jpg"> 图5:无激光和激光小鼠之间单核噬细胞的代表性数据，无论有没有系统性注水。MHCII- （A）， CD11c- （B） 和 CD11c+ （C） 巨噬细胞计数都增加了激光。未检测到无激光微胶质（D）或单核细胞（E）之间的任何变化。直流细胞（DC）也增加了激光（F）。布朗福赛斯和韦尔奇阿诺瓦与邓内特的T3多重比较测试被用来定义统计差异，由于不同群体之间的不平等差异。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61348/61348fig05large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

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. ...

digestion-whole-mouse-eyes-for-multi-parameter-flow-cytometric

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

4.8K 次观看.  Northwestern University. 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。

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

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

科研

神经科学

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and subsequently digests this material, commonly through lysosomal degradation. Microglia are the resident immune cells of the central nervous system (CNS) whose phagocytic function has been described in a broad range of conditions from neurodegenerative disease (e.g., beta-amyloid clearance in Alzheimer&#8217;s disease) to development of the healthy brain (e.g., synaptic pruning)1-6. The following protocol is an engulfment assay developed to visualize and quantify microglia-mediated engulfment of presynaptic inputs in the developing mouse retinogeniculate system7. While this assay was used to assess microglia function in this particular context, a similar approach may be used to assess other phagocytes throughout the brain (e.g., astrocytes) and the rest of the body (e.g., peripheral macrophages) as well as other contexts in which synaptic remodeling occurs (e.g. ,brain injury/disease).

Microglia are the resident immune cells of the central nervous system (CNS) with a high capacity to phagocytose or engulf material in their extracellular environment. Here, a broadly applicable, reliable, and highly quantitative assay for visualizing and measuring microglia-mediated engulfment of synaptic components is described.

一个吞噬含量:一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

评估视网膜小胶质细胞吞噬功能<em&gt;在体内</em&gt;使用基于流式细胞术流量测定

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

免疫与感染

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 ...

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and temporal specificity2. Chemotaxis is a critical function of lymphocytes and other immune cells that can be quantitatively assessed in vitro. This manuscript describes methods that permit the evaluation of chemotaxis, both in vitro and in vivo, for diverse cell types including cell lines and native cells. The in vitro, plate-based format permits the comparison of several conditions simultaneously in real-time, and can be completed within 1-4 h. In vitro assay conditions can be manipulated to introduce agonists and antagonists, as well as differentiate chemotaxis from chemokinesis, which is random movement. For in vivo trafficking assessments, immune cells can be labeled with multiple fluorescent dyes and used for adoptive transfer. The differential labeling of cells allows for mixed cell populations to be introduced into the same animal, thereby decreasing variance and reducing the number of animals required for an adequately powered experiment. Migration into lymphoid tissue occurs in as little as 1 h, and multiple tissue compartments can be sampled. Flow cytometry following tissue harvest allows for a rapid and quantitative analysis of the migratory patterns of multiple cell types.

Quantifying Human Monocyte Chemotaxis In Vitro and Murine Lymphocyte Trafficking In Vivo

Protocols for quantitative assessment of lymphocyte chemotaxis and migration are important tools for immunology research. Here, an in vitro protocol is described that permits real-time, multiplexed evaluation of cell migration, as well as a complementary in vivo technique enabling tracking of native cells to spleen.

人单核细胞趋化体外和小鼠淋巴细胞贩运体内

Chemotaxis is migration along a specific chemical gradient1. Chemokines are chemotactic cytokines that promote cellular trafficking with anatomic and ...

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

生物化学

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

小鼠巨噬细胞吞噬的延时3D 成像

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

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

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 lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population 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 ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.

增强型绿色荧光蛋白表达大肠杆菌评价小鼠腹腔巨噬细胞吞噬细胞增多

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a ...

<meta charset="utf8"></head><body>小鼠全支架视网膜中 RGC 的免疫染色和基于 AI 的计数

Whole-Mount Immunostaining and Automatic Counting of Mouse Retinal Ganglion Cells

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve as valuable animal models for studying the pathogenesis and treatment of glaucoma due to their relatively homogenous genetic background and retinal ganglion cells (RGCs), which structurally resemble those in humans. Accurate assessment of RGC damage and treatment outcomes in mouse glaucoma models requires determining the RGC number across the entire retina. This protocol outlines a comprehensive method involving the isolation of the whole retina, labeling RGCs with specific antibodies, and rapid, accurate automatic counting of RGCs using an AI-based program. The streamlined approach allows for efficient and precise quantification of RGC numbers in mouse retinas, facilitating the evaluation of RGC degeneration and potential therapeutic interventions. By enabling researchers to assess the extent of RGC damage, this protocol contributes to a deeper understanding of glaucoma pathogenesis and aids in developing effective treatment strategies to manage and prevent vision loss.

<meta charset="utf8"></head><body>作者聚焦:青光眼小鼠模型中的高效视网膜神经节细胞计数用于治疗评估

This protocol describes the procedure for isolating the whole-mount mouse retina and performing immunostaining to label all retinal ganglion cells (RGCs). The process is followed by imaging and automatically counting RGCs using AI-based software, providing a simple, fast, and accurate method for quantifying RGCs in the entire mouse retina.

小鼠视网膜神经节细胞的全支架免疫染色和自动计数

Glaucoma is a leading cause of blindness globally, characterized by a complex pathogenic mechanism that makes vision restoration challenging. Mice serve ...

全鼠眼的消化，用于单核噬细胞的多参数流细胞学分析

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整装免疫荧光染色新生小鼠视网膜血管生成调查