该研究项目由新加坡国立医学研究理事会（NMRC）提供的新研究者资助。该小组希望承认2012年11月至2014年10月在德国大学学院眼科研究所（IoO）向国立医学研究委员会（NMRC）海外研究培训奖学金提供的研究培训。大志志Agrawal博士获得了Shima博士实验室的细胞标记和活体成像的概念和技能。因此，团队希望在David Shima教授，Kenith Meissner教授，Peter Lundh博士和Daiju Iwata博士的培训研究中感谢监督和指导。

本研究中使用的动物方案由新加坡SingHealth的机构动物护理和使用委员会批准，并符合视力和眼科研究协会（ARVO）关于在眼睛和视力中使用动物的声明的指导原则研究。 荧光染料对红细胞和白细胞的标记<ol><li> 试剂的制备 <ol><li>通过将3mg的ICG溶解在1800μL无菌蒸馏水中来制备ICG（1.5mg / mL）。加入200μL10倍磷酸缓冲盐水（PBS）。 </li><li>通过向6 mL无菌蒸馏水中加入4 mL 1x PBS制备40％1x PBS。通过向10ml的1x PBS中加入100mg BSA，制备1％牛血清白蛋白（BSA）。充分混匀，直到BSA溶解。 </li></ol></li><li> 使用密度梯度离心分离红细胞和白细胞 <ol><li>麻醉老鼠（野蛮（C57BL / 6），使用氯胺酮盐酸盐（50mg / kg）和盐酸赛拉嗪（0.5mg / kg）（根据ARVO指南的时间表1）。通过踏板取反反射（ 即脚趾捏）确认麻醉。 </li><li>慢慢地将29号针头（连接到1 mL注射器）以针对心脏的朝向角度插入。要确认针是否在心脏内部，请稍微抽出柱塞，并检查是否将血液注入注射器。直接从心脏排出0.8 - 1 mL的血液。 </li><li>立即将血液转移到含有采血管的乙二胺四乙酸（EDTA）（1.8mg / mL血液）中，并轻轻混合以防止血液凝结。 </li><li>通过腹膜内注射施用戊巴比妥（80mg / kg）来安乐死小鼠。 </li><li>在2mL微量离心管和中心离心管中将全血覆盖在聚蔗糖和亚氨基酸钠溶液（1:1比例;密度1.077g / mL）的顶部fuge（450×g，30分钟）以允许从血浆中分离白细胞和红细胞。 </li><li>使用移液管取出并丢弃上清（血浆）。使用移液器小心地将血沉棕黄层（白细胞）和红细胞（红细胞层）转移到新的和分开的管中。 </li></ol></li><li> ICG（1.5％）标记红细胞 <ol><li>用1x PBS洗涤红细胞以除去任何污染物。将红细胞重悬于1×PBS（1:1）中以制备50％血细胞比容。将0.5mL 50％血细胞比容（〜250μL包装的红细胞）等分成微量离心管。离心红细胞（750×g，5分钟），弃去上清液。 </li><li>向沉淀的红细胞中加入200μL的40％1X PBS，混匀，室温孵育5分钟。向红细胞悬浮液中加入100μL的ICG（1.5mg / mL），通过倒置管轻轻混合，并在室温下孵育5分钟。加入300μL1x PBS，并孵育37℃，摇床培养箱中60分钟。 </li><li>离心载体红细胞（750 x g，3 min），并重悬于1 mL 1x PBS中。 </li><li>用1x PBS洗涤细胞〜3-5次，直到上清液清除（步骤1.3.3）。最后一次洗涤后，倾倒上清液，加入1％BSA PBS（1:1）至标记的预溶胀红细胞，以达到ICG标记红细胞的50％血细胞比容（1.25×10 8个细胞/ mL）。 </li></ol></li><li> 荧光素钠（1％）标记白细胞 <ol><li>使用密度梯度离心（从步骤1.2.6）将血沉棕黄层与血液分离后，用10ml的1x PBS洗涤细胞一次，通过450×g离心10分钟除去任何污染物。将沉淀重悬于900μL1x PBS中。 </li><li>将100μL10％的荧光素钠加入到900μL白细胞悬浮液中，并在室温下孵育2分钟。用10mL 1X PBS通过离心机洗涤标记细胞三次ng（450×g，10分钟）。将细胞重悬于100μL1x PBS中。 </li></ol></li></ol> 2.荧光标记细胞的实时成像<ol><li> ICG标记红细胞的活细胞成像 <ol><li>为了通过尾静脉或后视轨道途径制备注射细胞，用1x PBS将ICG标记的红细胞的50％血细胞比容稀释10倍和50倍，分别制备5％和1％的血细胞比容细胞。 </li><li>将鼠标置于红外光（250W强度）下5分钟，使尾静脉扩张。通过腹膜内注射麻醉氯胺酮盐酸盐（50mg / kg）和盐酸赛拉嗪（0.5mg / kg）（根据ARVO指南，按照时间表1）麻醉小鼠。 </li><li>用0.5％托品酰胺和2.5％盐酸去氧肾上腺素滴每眼瞳孔。 </li><li>将隐形眼镜放在一只眼睛上进行成像。使用眼科凝胶作为成像介质并防止干燥的角膜。用+25屈光度镜头固定共焦激光扫描检眼镜（SLO）相机，以补偿鼠标眼睛的折射。 </li><li>将鼠标放在SLO成像平台上。确保鼠标的角膜朝向SLO机器的光学头部。 </li><li>打开成像模块。使用相关的成像模块软件，点击"新病人"按钮，添加鼠标标签号，出生日期，荧光染料百分比和标记细胞类型等动物识别细节。 <ol><li>通过操纵成像模块将动物的视神经放置在成像屏幕的中心。使用相关的成像模块软件，单击"获取"按钮以30°或15°角度视图设置拍摄红外（IR）眼底图像。确保视网膜的中心，瞳孔和激光的光路对齐，以获得最佳质量的图像。 <br/&gt;注意:通过操纵操纵杆来对准光路，以获得最清晰的图像和最佳的照明曝光。 </li></ol></li><li>采取ICG眼底视频。 <ol><li>打开ICG滤镜（790 nm），将相机设置为30°或15°角度视图的高速模式，并将所有读数的ICG强度设置为85，以进行标准化。在控制面板上，点击"获取"按钮，以8.8 / 15帧/秒的速度拍摄视频（基线控制）1分钟。 </li></ol></li><li>将100μLICG标记的红细胞加载到连接到30号针的胰岛素注射器中。 <ol><li>对于通过尾静脉注射进行注射，将针头斜面插入尾静脉。通过检查进入注射器的血液回流来确认静脉通路，并将标记的细胞注入循环。 </li><li>对于通过后视轨道进行注射，将眼睛附近的针插入后视镜间隙即通过检查血液进入注射器的回流并将标记的细胞注入循环来确认后视眼通路。 </li></ol></li><li>注射后，重新定位鼠标，并使用ICG过滤器（见步骤2.1.7.1）获取IR眼底图像（参见步骤2.1.6.1）和视频。视网膜循环中的标记细胞可在注射细胞后立即显现。 </li><li>获取视频帧后，使用SLO查看模块软件导出视频时，通过选择.tiff格式来提取视频序列的图像。将文件保存在所需的文件夹中。 </li><li>取下隐形眼镜，将其放在另一只眼睛上，按照步骤2.1.4 - 2.1.7所述完成成像。 </li><li>成像后，将麻醉动物置于红外光（250W）下，以维持体温，直到从麻醉中恢复。当动物完全康复时，将鼠标放回到保持架中。</li></ol></li><li> 1％荧光素钠标记白细胞的活细胞成像 <ol><li>按2.1节所述准备鼠标并安装仪器。 </li><li>按照步骤2.1.4 - 2.1.6中的步骤放置鼠标并拍摄红外眼底图像。 <ol><li>使用荧光素过滤器（488 nm），使用30°或15°角度视野的高速摄像模式和85°的荧光强度来获取所有读数的小鼠的荧光素血管造影。录制视频（基线控制）为8.8 / 15帧/秒（见步骤2.1.7.1）。 注意:在这里，以不同的时间间隔获取了多个视频轨道（1分钟视频）。 </li></ol></li><li>通过尾静脉或后眼轨注射将100μL标记的白细胞注入小鼠，如2.1.8节所述。 </li><li>注射后立即放置鼠标并取出红外眼底图像（参见步骤2.1.5和2.1.6）和荧光小鼠的再凝血血管造影（见2.2.2节）。 </li><li>通过转动成像模块上的对焦旋钮调整扫描激光的焦点，观察视网膜或脉络膜血管中的标记细胞。 </li><li>使用SLO查看模块软件获取视频帧和图像（参见步骤2.1.10）并将其保存在所需的文件夹中。 </li><li>手术结束后，按照步骤2.1.12处理动物。 </li></ol></li><li> MtrackJ软件进行图像分析 <ol><li>通过点击"文件"打开ImageJ中的图像序列。选择"导入"并选择"图像序列"，选择所需的图像文件夹，然后单击"打开"。屏幕上将出现一个名为"序列选项"的弹出窗口。点击"确定";图像序列将打开。 </li><li>通过点击"图像"设置图像视频的帧间隔，并选择"属性"进行速度测量。这里是8.8框ES /秒。 </li><li>通过选择"插件"打开MTrackJ插件。选择"跟踪"，然后选择"MtrackJ"。应该会出现一个菜单。 </li><li>要调整跟踪设置，请选择"跟踪"（菜单下），并选中"添加点后移动到下一帧时间索引"框。 </li><li>跟踪第一个单元格。 <ol><li>找到一个单元格并跟踪后续帧中的相同单元格。在菜单上单击"添加"添加新的轨道。 </li><li>将光标悬停在图像上（+号），然后单击相应的单元格。 注意:MTrackJ将自动跳转到下一个时间范围，并添加一个小圆圈，标记轨迹中第一个点的位置。 </li><li>在移动通过图像序列的帧时，继续单击单元格的当前位置。 注意:偶尔，标记轨迹上的点的圆圈会影响查看当前位置的能力。如果发生这种情况，进入步骤2.3.5.3.1。 <ol><li>通过单击菜单中的"显示"并选择"仅显示当前时间的轨迹点"来更改轨迹的显示选项。要查看所有轨迹，请取消选择此选项。 </li></ol></li><li>继续点击直到看到第一个单元格的轨迹。然后点击"保存"保存轨迹。 </li></ol></li><li>跟踪第二个单元格。 <ol><li>在菜单中，单击"添加"开始新的轨道。重复步骤2.3.5.1 - 2.3.5.4跟踪新单元格。点击"保存"保存第二个轨道。 </li><li>对于观察到的所有可跟踪单元，请继续执行这些步骤。跟踪单元格后，单击菜单选项卡中的"测量"和"确定"以获取结果。 注意:结果窗口自动打开，可以保存并打开电子表格进行进一步分析。 </li></ol></li></ol></li></ol>

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研究视网膜和脉络膜循环中的血液动力学对于了解许多眼部疾病的病理生理学至关重要。视网膜循环中的血流动力学可以通过傅立叶域光学相干断层扫描（FD-OCT），激光散斑图（LSFG）和视网膜血氧饱和度检测来研究。尽管这些方法使用不同的方法来研究视网膜循环40,41,42,43,44,45中的总血流量，但是它们具有不能研究各种细胞类型的流动动力学的限制。视网膜和脉络膜组织的营养物质和氧气供应以及免疫细胞在眼部炎症疾病中的迁移及其在疾病发病机理中的作用可以通过调查呃的流动动力学来研究分别在视网膜和脉络膜循环中的红细胞和白细胞。与成像方法相结合的标记技术将有助于跟踪循环中的标记细胞并研究标记细胞的流动动力学。成功应用了几种荧光染料来标记白细胞和红细胞，以调查它们在循环中的流动动力学3,17,18,19。 在这里，我们报告食品和药物管理局批准的无毒荧光染料，吲哚菁绿和荧光素钠的应用，分别用于标记和观察视网膜循环中的红细胞和白细胞。虽然荧光素钠是用于跟踪白细胞31,32,33的最常用的染料<s， 34 ，分别用ICG和荧光素钠同时标记红细胞和白细胞的优点分别是通过改变SLO中的滤膜在动物眼睛中同时有效地观察标记细胞。 SLO是视网膜成像的非侵入性方法，广泛用于人类和动物中各种视网膜疾病病理变化的研究。它也可以帮助比较视网膜变化对给定治疗的反应46 。 为了在SLO中获得高质量的图像，瞳孔需要充分扩张，必须使用眼科凝胶使隐形眼镜保持湿润的角膜并防止白内障形成。眼睛的位置和相机的光路必须是直线的，以获得优质的眼底和血管造影图像。动物应深入镇静，以尽量减少成像期间的运动。尽管ICG的1％和5％血细胞比容均为1.5％标记的红细胞表现出视网膜循环中荧光标记细胞的可视化，1％血细胞比容更适合于监测各个细胞。如果注射的白细胞数量太低，荧光标记的细胞就不能显现。因此，为了在监测视网膜循环中的标记细胞方面取得更好的效果，建议从至少4只小鼠收集白细胞，用1％的荧光素钠标记，并注射到单个小鼠中。尽管荧光素钠（钙黄绿素，FITC，DIO，FDA和CFDA具有类似于荧光素钠或荧光素附近的吸收/发射最大值）的替代染料，但染色保留率和对眼组织的毒性必须评估为9,12,13 ， 14，17 。 报道的技术的限制是60分钟后荧光信号的循环衰减。因此，必须在60分钟内分析标记细胞的流动动力学。在这里，为了标记白细胞，从一只小鼠中取出血液，将细胞分离，标记，注射到另一只小鼠中，并通过SLO观察。然而，在SLO中，在视网膜血管中仅观察到每帧的0-1个细胞。这可能是由于白细胞数量不足，因此我们建议从至少3-4只小鼠采集血液以显现每帧较高的细胞数。在研究中使用了8.8帧/秒的相机速度，但是可以增加这种速度以实现更多连续的标记细胞的图像和视频。 早期研究报道了DR不同阶段的糖尿病患者视网膜循环血流速度变化。 Clermont 等人报道了DR 47早期患者视网膜血流减少33％，Nguyen 等人。报道了具有增殖性DR 48的糖尿病患者的视网膜血流量增加。据报导，血流速度降低与进行性青光眼49有关 。在上述研究中，作者调查了视网膜循环中的全血流速度。研究每种细胞类型的流速可以提供关于不同疾病和不同疾病阶段的细胞相互作用的信息，并且可以帮助预后。在这里我们报告一种方法来分别用ICG和荧光素钠标记红细胞和白细胞，并在视网膜循环中可视化;该方法可应用于任何疾病模型进行体内研究。 未来的研究可以利用我们的标签技术使用SLO来研究不同药物治疗条件下标记的红细胞和白细胞的流动动力学变化在疾病的各个阶段。此外，我们的方法可以帮助了解红细胞和白细胞在疾病表型中的作用。

了解视网膜和脉络膜循环中血液细胞的流动动力学对于了解潜在的威胁眼睛的眼部疾病和其他眼部炎症状况的发病机制至关重要。然而，涉及荧光染料与血浆蛋白结合的常规血管造影技术不提供关于红细胞或白细胞1的动力学的任何信息。红细胞视网膜流动力学对于研究视网膜中的代谢有效循环和白细胞流动动力学是重要的，用于了解各种炎性疾病中的细胞迁移，识别，粘附和破坏2 。用于鉴定和表征各种细胞类型的几种荧光分子3 。血细胞的血液动力学可以用适当的荧光染色来测量适用的成像技术4 。 眼内疾病如年龄相关性黄斑变性（AMD）和糖尿病性视网膜病变（DR）中炎症反应的存在涉及淋巴细胞在患病区域5,6的积累。跟踪组织中的免疫细胞可以帮助了解涉及疾病发病机制的复杂事件。放射性同位素如51 Cr和125 I在早期研究中被用作细胞示踪剂。这些染料是有毒的并影响细胞活力。虽然放射性标记物3 H和14 C对细胞的毒性较小，但由于其发射能量较低，因此难以在系统7,8中检测到信号。引入了许多荧光染料以克服与w相关的潜在问题使用荧光显微镜和流式细胞术，体外放射性标记和轨道淋巴细胞迁移9,10 。 Hoechst 33342和噻唑橙是DNA结合荧光染料，用于在体内追踪淋巴细胞。 Hoechst 33342结合DNA中富含AT的区域，是膜可渗透的，保留荧光信号2 - 4天，耐猝灭9,10 。 Hoechst 33342和噻唑橙的缺点分别是抑制淋巴细胞增殖11和短半衰期9 。 钙黄绿素AM，荧光素二乙酸酯（FDA），2&#39;，7&#39;-双 - （2-羧乙基）-5-（和-6） - 羧基荧光素，乙酰氧甲基酯（BCECF-AM），5-（和-6）羧基荧光素二乙酸酯（CFDA）和5-（和-6） - 羧基荧光素二乙酸酯乙酰氧甲酯（CFDA-AM）是用于淋巴细胞迁移研究的细胞质荧光染料。然而，FDA，CFDA和CFDA-AM在细胞中的保留率较低9 。 BCECF-AM降低增殖反应并影响趋化性和超氧化物的生成9,12 。钙黄绿素AM是一种荧光染料，可用于短期体内淋巴细胞迁移研究。它发出强烈的荧光信号，不干扰大多数细胞功能，并保留荧光信号长达3天12,13 。荧光素异硫氰酸酯（FITC）和羧基荧光素二乙酸酯琥珀酰亚胺酯（CFDA-SE）是共价偶联荧光染料，用于淋巴细胞迁移研究。 FITC对细胞活力没有影响，与B淋巴细胞的亲和力比T淋巴细胞14,15更强</suP&gt;。 CFDA-SE标记的淋巴细胞可以在体内追踪超过8周和最多8个细胞分裂9,16 。 C18 DiI（1,1&#39;-二十八烷基-3,3,3&#39;，3&#39;-四甲基吲哚羰花青高氯酸盐），DiO（3,3&#39;-二十八烷基羰基花青高氯酸盐），Paul Karl Horan（PKH）2，PKH3和PKH26是膜 - 插入用于标记白细胞和红细胞的荧光亲脂性碳菁染料。 C18 Dil和DiO在掺入细胞膜时表现出较高的信号，并且相对无毒12,17 。 PKH2，PKH3和PKH26标记的细胞表现出良好的荧光信号保留，毒性较小18,19,20,21,22。然而，PKH2下调CD62L表达并减少<html>细胞活力23 。 大多数上述研究已经被执行用于跟踪淋巴细胞迁移和淋巴管中的增殖并研究非眼部循环中标记的红细胞。使用标记技术研究眼部循环中的血细胞的研究很少。扫描激光检眼镜（SLO）的应用在通过眼底血管造影技术研究体内视网膜和脉络膜循环中的标记细胞方面具有很大的优势。有几种荧光染料，例如ICG，吖啶橙，FITC，荧光素钠和CFDA，用于研究SLO 25,26,27,28,29,30,30在视网膜循环中的白细胞。class ="xref"&gt; 31，32，33，34。吖啶橙26,27的光毒性和致癌性，FITC与细胞活性的干扰以及血管内造影剂对视网膜和脉络膜血管分辨率的要求限制了其在体内动物实验中的应用29 。荧光素钠和ICG无毒，经食品和药物管理局批准，可安全用于人类测试32,35 。大多数流动动力学研究与白细胞或红细胞的标记及其在视网膜和脉络膜血管中的可视化有关36,37,38,39 </s了&gt;。在这里，我们描述了红细胞ICG标记的标准化方案，白细胞的荧光素钠标记，以及使用SLO跟踪小鼠视网膜循环中的可视化标记细胞。

<table><tbody><tr><td>Cardiogreen polymethine dye (Indocyanine green)</td><td>Sigma Aldrich</td><td>12633-50MG</td><td></td></tr><tr><td>Fluorescein 100 mg/mL</td><td>Novartis</td><td>U1705A/H-1330292</td><td></td></tr><tr><td>10x Phosphate-buffered saline (PBS) Ultra Pure Grade</td><td>1st BASE</td><td>BUF-2040-10X1L</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A7906-100G</td><td></td></tr><tr><td>Microtainer tubes with K2E (K2EDTA) - EDTA concentration - 1.8 mg/mL of blood</td><td>BD, USA</td><td>REF 365974</td><td></td></tr><tr><td>Histopaque 1077 solution</td><td>Sigma Aldrich</td><td>10771</td><td></td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf</td><td>05-413-401</td><td></td></tr><tr><td>Microcentrifuge tubes 2 mL</td><td>Axygen</td><td>MCT-200-C-S</td><td></td></tr><tr><td>Vortex mixer</td><td>Insta BioAnalytik pte. ltd</td><td>FINE VORTEX</td><td></td></tr><tr><td>Shaker incubator</td><td>Lab Tech</td><td></td><td></td></tr><tr><td>Ceva Ketamine injection (Ketamine hydrochloride 100 mg/mL)</td><td>Ceva</td><td>KETALAB03</td><td></td></tr><tr><td>ILIUM XYLAZIL-20 (Xylazine hydrochloride 20 mg/Ml)</td><td>Troy Laboratories PTY. Limited</td><td>LI0605</td><td></td></tr><tr><td>1% Mydriacyl 15 mL (Tropicamide 1%)</td><td>Alcon&amp;nbsp;Laboratories, Inc. USA</td><td>NDC&amp;nbsp;0998-0355-15</td><td></td></tr><tr><td>2.5% Mydfrin 5 mL (Phenylephrine hydrochloride 2.5%)</td><td>Alcon&amp;nbsp;Laboratories, Inc. USA</td><td>NDC&amp;nbsp;0998-0342-05</td><td></td></tr><tr><td>Terumo syringe with needle 1 cc/mL Tuberculin</td><td>Terumo (Philippenes) Corporation, Philippines</td><td>SS-01T2613</td><td></td></tr><tr><td>Vidisic Gel 10 G</td><td>Dr. Gerhard Mann, Chem.-Pharm, Fabrik Gmbh, Berlin, Germany</td><td></td><td></td></tr><tr><td>Alcohol swabs</td><td>Assure medical disposables</td><td>7M-004-L-01</td><td></td></tr><tr><td>Confocal laser scanning angiography system (Heidelberg Retina Angiograph 2)</td><td>Heidelberg Engineering, GmbH, Heidelberg, Germany</td><td></td><td></td></tr><tr><td>Hiedelberg Spectralis Viewing Module software, v4.0</td><td>Heidelberg Engineering, GmbH, Heidelberg, Germany</td><td></td><td></td></tr><tr><td>Fluorescent microscope</td><td>ZEISS</td><td>Model: axio imager z1</td><td></td></tr></tbody></table>

fluorescent dye labeling of erythrocytes and leukocytes for studying the flow dynamics in mouse retinal circulation

视网膜和脉络膜血流动力学可以提供各种眼部疾病如青光眼，糖尿病性视网膜病变，年龄相关性黄斑变性（AMD）和其他眼部炎症病症的病理生理学和后遗症的见解。它也可能有助于监测眼睛的治疗反应。血细胞的适当标记加上标记细胞的活细胞成像，允许调查视网膜和脉络膜循环中的流动动力学。在这里，我们分别描述1.5％靛青绿（ICG）和1％荧光素钠标记小鼠红细胞和白细胞的标准化方案。应用扫描激光检眼镜（SLO）可视化C57BL / 6J小鼠（野生型）视网膜循环中的标记细胞。两种方法都在小鼠视网膜循环中显示了不同的荧光标记细胞。这些标记方法可以在各种眼部疾病中具有更广泛的应用楷模。

用1.5％ICG标记的红细胞在C57BL / 6J小鼠（野生型）的视网膜循环中显现。 1.5％ICG标记的红细胞的1％和5％血细胞比容在视网膜循环中是可区分的。然而，用1％红细胞比容为1.5％的ICG标记的红细胞，可以更清楚地观察到各个标记细胞的可视化（ 图1 ）。在5％的血细胞比容中，由于视网膜血管中的标记细胞数量很多，不可能标记个体细胞。在两个条件下，在周围毛细血管区域观察到一些红血球，但在标记的红细胞的5％血细胞比容中更突出（ 图2 ）。 按照上述方案，用1％的荧光素钠成功标记白细胞，并使用荧光显微镜将其显示为绿色标记细胞<strong<html> class ="xfig"&gt;图3）。在标记的样品中也观察到小块细胞（4〜5个细胞在一起）。当细胞注射到小鼠循环中时，标记的白细胞在视网膜循环中可视化（ 图4 ）。标记后，将红细胞和白细胞立即注射到小鼠中，并以30分钟和60分钟的时间间隔显现。在注射后立即观察荧光标记的细胞，但荧光强度以30分钟和60分钟的时间间隔逐渐降低。我们还使用白细胞染色方案（1％荧光素钠）在红细胞上，并且没有观察到红细胞的标记。在Image J公共领域软件上使用Mtrack J插件，在视网膜循环中跟踪细胞（ 图5 ）。 95 / 55495fig1.jpg"/&gt; 图1 :视网膜循环中1.5％ICG标记的红细胞的1％血细胞比容。 ICG标记的个体红细胞在视网膜血管中显示明亮的信号。在周围毛细血管区域观察到一些红血球。刻度=200μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55495/55495fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55495/55495fig2.jpg" /> 图2 :视网膜循环中1.5％ICG标记的红细胞的血细胞比容为5％。许多ICG标记的红细胞在视网膜血管中显示明亮的信号;在周围毛细血管区域观察到大量的红血球。刻度=200μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55495/55495fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55495/55495fig3.jpg" /> 图3 :1％荧光素标记的白细胞荧光显微镜图像。荧光素标记的白细胞显示绿色荧光信号，小团块（放大20倍;标度=50μm）。 <a href="http://ecsource.jove.com/files/ftp_upload/55495/55495fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55495/55495fig4.jpg" /> 图4 :1％荧光素标记的白细胞在视网膜循环中的表达。荧光素标记的白细胞（箭头）在视网膜血管中显示明亮的信号。刻度=200μm。 <a href="http://ecsource.jove.com/files/ftp_upload/55495/55495fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图5" class="xfigimg" src="/files/ftp_upload/55495/55495fig5v2.jpg" /> 图5 :使用图像分析软件跟踪标记的单个细胞在视网膜循环中。测量不同的轨迹（彩色圆圈和图像上所示的线），以计算视网膜循环中标记细胞的平均速度。

Watch this Scientific Journal Video about Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation at JoVE.com

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

眼睛循环中标记血细胞的活细胞成像可以提供关于糖尿病视网膜病变和年龄相关性黄斑变性的炎症和缺血的信息。描述了标记血细胞并在视网膜循环中成像标记细胞的方案。

用于研究小鼠视网膜循环中流动动力学的红细胞和白细胞的荧光染料标记

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

fluorescent-dye-labeling-erythrocytes-leukocytes-for-studying-flow

Nanyang Technological University

Research

JoVE Journal

Medicine

12.4K 次观看.  Tan Tock Seng Hospital. 眼睛循环中标记血细胞的活细胞成像可以提供关于糖尿病视网膜病变和年龄相关性黄斑变性的炎症和缺血的信息。描述了标记血细胞并在视网膜循环中成像标记细胞的方案。 

视频: Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

活体鼠脑微循环显微镜使用一个封闭的颅窗口

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

科研

神经科学

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

癌症研究

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

视网膜血流小鼠活体视频显微镜测量

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

医学

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

免疫与感染

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, ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

一个小鼠红细胞亚群富集去核活动由多光谱成像流式细胞仪鉴定

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

生物学

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

鼠标心脏中毛细血管、动脉和腹膜的动态测量和成像

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

使用打开的血管窗口和活体荧光显微镜测量小鼠耳蜗中的心室血流

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

监测氧诱导视网膜病变小鼠模型中视网膜血管的动态生长

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...