这项研究由加拿大卫生研究院 (卫生研究院) 运营赠款 MOP-123419, 加拿大自然科学和工程研究委员会 418194, 以及安大略研究和创新部早期研究奖资助。DGW 贡献了一些图象, 对协议的优选和原稿的创作;他的资金来自利物浦大学的一个泵浦资助。Vanier 研究生奖学金和卫生研究院博士奖学金。供资机构在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。

来自健康志愿者的血液收集被西安大略大学健康科学研究伦理学委员会批准。静脉穿刺是根据三局人类研究政策声明的指引进行的。
1. THP-1 单核细胞系的培养和制备
<ol><li>培养 THP-1 单核细胞作为悬浮培养在 T25 烧瓶在37°c + 5% CO2。细胞应该生长在5毫升的罗斯威尔公园纪念研究所 1640 (RPMI 1640) + 10% 胎牛血清 (血清)。</li>
	<li>
每天通过轻轻摇动烧瓶, 将细胞均匀地悬浮在整个生长介质中, 然后立即用 hemocytometer 计数细胞。细胞密度达到 1 x 106细胞/毫升时应传代;
	<ol>
<li>预热 RPMI 1640 + 10% 在37°c 水浴中的血清。</li>
		<li>将 2 x 105细胞转化为1.5 毫升的离心管或15毫升圆锥管, 并在室温下离心 500 x g 的颗粒细胞为5分钟。</li>
		<li>除去上清液, 不干扰细胞颗粒, 并用重悬1毫升 (1.5 毫升离心管) 或5毫升 (15 毫升圆锥管) 的磷酸盐缓冲盐水 (PBS) 的颗粒。</li>
		<li>在室温下离心管 500 x 克, 在室温下为5分钟. 取出 PBS 而不干扰细胞颗粒。</li>
		<li>并用重悬颗粒在1毫升新鲜 RPMI 1640 + 10% 血清。</li>
		<li>入一个新的 T25 瓶地方4毫升温暖的媒介, 并且对此增加悬浮细胞从1.2.5。文化在37°c + 5% CO2孵化器, 直到细胞需要传代 (通常3天), 或直到细胞是需要的实验。</li>
	</ol>
</li>
	<li>
对于 THP-1-derived 巨噬细胞的实验, 在传代之前从烧瓶和盘子中取出所需的细胞数:
	<ol>
<li>将所需数量的18毫米圆形玻璃盖玻片 (#1 5 厚度) 放入12井板的井中-通常为1盖玻片每个条件和/或 timepoint。入每个井整除 5 x 104 THP-1 单核细胞。如果需要, 可以更改每个井中添加的单元格数。</li>
		<li>使用 RPMI + 10% 的血清加热到37摄氏度, 使每个细胞的总体积达到1毫升。</li>
		<li>添加 100 nM 佛波 12-酯 13-醋酸盐 (PMA) 的每一个井和培养3天, 以诱导分化的 THP-1 单核细胞成巨细胞样。</li>
	</ol>
</li>
</ol>2. J774.2 巨噬细胞系的培养和制备
<ol><li>
培养 J774.2 细胞在 T25 瓶在37°c + 5% CO2。细胞应该生长在5毫升的 Dulbecco 的改良鹰培养基 (DMEM) + 10% 血清和传代一旦文化达到80-90% 融合。通道单元格:
	<ol>
<li>从烧瓶中取出所有介质, 用5毫升 PBS 一次上升一次。</li>
		<li>从烧瓶中取出 PBS, 用5毫升新鲜的 DMEM + 10% 的血清代替。</li>
		<li>使用电池刮刀, 刮掉瓶子底部, 以暂停介质中的细胞。用力地将细胞移去几次, 以分解任何细胞聚集物。</li>
		<li>稀释细胞1:5 通过去除4毫升细胞悬浮从烧瓶和替换它与4毫升新鲜的媒介。剩余的细胞悬浮可以被丢弃, 用于在新鲜的 T25 瓶中启动新的细胞培养, 或用于实验。</li>
	</ol>
</li>
	<li>
使用 J774.2 细胞建立 efferocytosis 化验:
	<ol>
<li>在实验开始前的某一天, 按照步骤 2.1, 将 J774.2 细胞悬浮在5毫升的新鲜介质中. 1–2.1 3。用 hemocytometer 计数细胞, 并在浓度为 5 x 104细胞/毫升时准备必要体积的细胞。</li>
		<li>将必要数量的18毫米圆形玻璃盖玻片 (#1 5 厚度) 放入12井板井中。入每个井整除1毫升 5 x 104细胞或毫升细胞悬浮。</li>
		<li>文化过夜, 让细胞坚持盖玻片和恢复从传代。</li>
	</ol>
</li>
</ol>3. 原发性人 M2 巨噬细胞的培养
<ol><li>收集10毫升的血气人血液为每 12 M2 巨噬细胞需要。</li>
	<li>在15毫升离心管中, 5 毫升的人血在5毫升前预热 Lympholyte-聚细胞分离培养基上。准备多管, 如果处理 > 5 毫升的血液, 而不是使用较大的容积管。</li>
	<li>离心机在 300 x g 35 分钟, 使用中等加速度和无断裂。</li>
	<li>使用塑料 pipettor 小心地去除上单核细胞丰富的带, 并转移到50毫升离心管。如果在步骤3.2 中制备了多个管, 则带可以汇集成一个50毫升的管。带 PBS 的管容积可达50毫升。</li>
	<li>离心机以 300 x g 为8分钟, 取出上清液。在这一步的地方蒸压18毫米直径圆形盖玻片 (#1 5 厚度) 到每一个12井板井。</li>
	<li>并用重悬细胞颗粒在300µL 无血清 RPMI 1640 每期望的井数;例如,如果10毫升的血液被处理, 以准备一个完整的12井板, 悬浮细胞颗粒3.6 毫升的介质。</li>
	<li>将细胞悬浮液的300µL 添加到 12-井板中的每一个含盖玻片的井中。孵育1小时在37°c + 5% CO2。</li>
	<li>用1毫升温暖的 PBS 轻轻地清洗盖玻片 3x, 去除任何非黏附细胞。</li>
	<li>添加1毫升的 RPMI 1640 + 10% 血清 + 10 ng/毫升重组人脑脊液 + 细胞培养抗生素/防霉。孵育在37°c + 5% CO2 5 天。</li>
	<li>用 RPMI 1640 + 10% 血清 + 10 ng/毫升重组人脑脊液 + 10 ng/毫升重组人 IL-4 + 细胞培养抗生素/防霉取代培养基。孵化在37°c + 5% CO2 2 天, 以完成 M2 极化。</li>
	<li>极化巨噬细胞应在未来3天内使用。</li>
</ol>4. 凋亡 Jurkat 细胞的制备
<ol><li>培养 Jurkat 细胞在5毫升的 RPMI 1640 + 10% 血清在37°c + 5% CO2。Jurkats 是悬浮细胞系, 每3–5天可由传代1:5 保持为新鲜、预热的介质。</li>
	<li>为制备凋亡细胞, 允许 Jurkat 培养成高密度 (传代后第4-5 天)。整除1.5 毫升到1.5 毫升离心管和颗粒细胞通过离心在 500 x g 5 分钟。</li>
	<li>在1毫升无血清 RPMI 1640 培养基中, 放弃上清和再悬浮细胞颗粒, 含1µM staurosporine。</li>
	<li>孵育16小时在37°c + 5% CO2使细胞凋亡。</li>
	<li>
如果需要, 确认诱导细胞凋亡的染色与蛋白 V:
	<ol>
<li>整除100µL staurosporine 处理 Jurkat 细胞培养成1.5 毫升离心管。颗粒细胞通过离心在 500 x g 5 分钟, 放弃上清, 并重新悬浮细胞颗粒在100µL 无血清 RPMI 1640 培养基。</li>
		<li>添加1µL 荧光素异硫氰酸酯 (FITC)-共轭蛋白 V, 在室温下孵育10分钟。</li>
		<li>添加900µL 的 PBS, 并将整个体积转移到一个12井板, 其中包含一个18毫米圆形玻璃盖玻片 (#1 5 厚度) 的单井。在装有 200 x g 的平板适配器的离心机上旋转, 以1分钟的力迫使细胞坚持盖玻片。或者, 可以将单元格放置到腔内幻灯片中进行成像。</li>
		<li>图像使用荧光显微镜。</li>
	</ol>
</li>
</ol>5. 使用固定细胞 Efferocytosis 测定和内出染色量化 Efferocytic 摄取和动力学
<ol><li>分别在1-3 节中描述 THP-1、J774.2 或 M2 人巨噬细胞。</li>
	<li>在实验开始前的晚上, 准备凋亡 Jurkat 细胞, 如4 节所述。</li>
	<li>实验前立即用 hemocytometer 计数凋亡细胞。将足够数量的凋亡细胞转化为1.5 毫升离心管-我们通常添加 5 x 105细胞/好, 提供一个目标: efferocyte 比率为10:1。</li>
	<li>颗粒 Jurkat 细胞通过离心在 500 x g 5 分钟和并用重悬 500 ul 的 PBS。</li>
	<li>在离心过程中, 整除10µL 的亚砜为新的1.5 毫升离心管。溶解到亚砜中的 hydroxysuccinimidobiotin (NHS-生物素) 的极小量。5-10 水晶 (~ 0.005 毫克) 是充足的。</li>
	<li>将 500 ul 凋亡细胞/PBS 悬浮液转移到亚砜/NHS-生物素含管。然后稀释细胞跟踪染料到制造商的建议浓度到凋亡细胞悬浮。一定要选择一个细胞跟踪染料, 不重叠的光谱与 FITC-链亲和素 (如红色或远红色细胞跟踪染料)。</li>
	<li>在昏暗的室温下孵化20分钟的悬浮液。添加等量的 RPMI 1640 + 10% 的血清和孵化5分钟在室温下, 以淬火任何反应染料。</li>
	<li>颗粒细胞通过离心在 500 x g 5 分钟, 放弃上清, 并在100µL RPMI 1640 + 10% 的巨噬细胞中重新悬浮染色的凋亡细胞。</li>
	<li>在巨噬细胞中添加100µL 染色凋亡细胞悬浮液滴状。离心机 200 x g 为1分钟的离心机装有一个板适配器, 以迫使接触巨噬细胞和凋亡单元。</li>
	<li>孵育板材为期望期间在37°c + 5% CO2在组织培养孵化器。对于巨噬细胞, efferocytosed 物质通常在20–30分钟后最先检测到, 并在120–180分钟后完成。</li>
	<li>在所需的时间点 (s) 从孵化器中移除单元格。用1毫升室温 PBS 冲洗细胞, 以停止 efferocytosis 和去除非 efferocytosed 凋亡细胞。</li>
	<li>添加 FITC 共轭链亲和素在 1:1, 000 稀释到每个井和孵化20分钟在黑暗中。这将标记暴露的生物素对任何非 efferocytosed 凋亡细胞材料。 注: 如果需要, 细胞细胞核可以在这一步骤染色, 添加1:20,000 稀释赫斯特33342或 4 ', 6-Diamidino-2-苯基吲哚, 盐酸盐 (DAPI)。</li>
	<li>用1毫升 PBS 冲洗细胞3次, 轻轻摇动或摇动样品, 每冲洗5分钟。将4% 多聚甲醛 (粉煤灰) 的细胞固定在 PBS 中, 室温下20分钟。用 PBS 冲洗细胞以去除过量的粉煤灰。</li>
	<li>在幻灯片上安装盖玻片以进行成像和转移到荧光显微镜。捕获有足够数量的单元格的 z 栈, 用于精确量化--通常10–30细胞的每个条件。非内化凋亡细胞材料将在产生的图像中明显显示为细胞跟踪染料标记的材料包裹的 FITC-链亲和素染色, 而 efferocytosed 材料形成离散细胞跟踪染料 puncta 无任何链亲和素染色。</li>
	<li>通过多种措施量化 efferocytosis 在产生的图像中的数量:
	<ol>
<li>通过确定每个巨噬细胞的离散 efferosomes (单元跟踪染料+/链亲和素 puncta) 的平均数量, 计算 efferocytic 指数。仅量化绑定到凋亡细胞或含有≥1可见 efferosomes 的巨细胞。记录巨噬细胞绑定到凋亡单元, 但缺乏离散 efferosomes, efferocytic 指数为0。</li>
		<li>通过测量含有≥1 efferosome 的巨噬细胞的分数来计算 efferocytic 效率。</li>
		<li>在多个时间点上固定和染色的成像细胞计算 efferocytosis 的速率。只有图像巨噬细胞被绑定到凋亡, 或包含可见的 efferosomes, 与 z 栈捕获的每个细胞。一旦成像, 确定 efferocytosis 的速率:<ol>
<li>使用链亲和素染色作为指南, 和在斐济/ImageJ26 (或其他图像分析软件包) 的手绘或多边形选择工具, 循环所有的细胞跟踪染料+/链亲和素-(如内化)材料在一个 z 栈的单平面。测量细胞跟踪染料这个区域的综合强度。每个 efferosome (如直径、相对于细胞边界或原子核的定位等) 的个别度量值15、27可以很容易地与这些测量同时获得, 大大增加分析过程中收集的数据。</li>
			<li>在同一 z 节上, 使用链亲和素染色作为指南和手绘或多边形选择工具, 将所有的细胞跟踪染料+/链亲和素+ (如非内化) 材料在一个 z 栈的单平面上循环.仅包括与吞噬接触的凋亡细胞的染色。测量该区域的综合强度。</li>
			<li>对图像的其余 z 部分重复步骤4.2.3.1 到4.2.3.2。总结了 efferocytosed (σ) 和非 efferocytosed(σ) 材料的综合强度.细胞凋亡细胞的 efferocytosed 可以计算为: <img alt="Equation" src="/files/ftp_upload/58149/58149eq1.jpg">
</li>
			<li>在所有时间点量化所有单元格的分数 efferocytosed。请注意, 凋亡细胞/efferosomes 的荧光强度可能因单个凋亡细胞摄取细胞追踪染料的变化而异, 并由于图像采集参数如曝光时间的变化而不同。因此, 只比较规范化的值, 如分数 Efferocytosed, 或与强度无关的值, 如 efferocytic 指数和 efferocytic 效率, 图像之间, 实验条件之间, 重复实验。</li>
		</ol>
</li>
		<li>为了确保结果数据集准确地反映细胞之间 efferocytosis 的变化, 对每个实验中可能的最大细胞数进行分析。 注: Efferocytic 效率和 Efferocytic 指数可以快速计算 (几秒钟/细胞), 我们通常的目的是分析每实验至少100个细胞, 重复每个实验至少3次。计算 efferocytosed 材料的分数, 和 efferosome 特定的措施更费力, 通常采取2–3分钟/细胞的经验丰富的分析师。对于这些计算, 我们每一个条件, 每个实验的最小15细胞。</li>
		<li>在每个单元格上记录 efferocytic 索引、分数 efferocytosed 和单个 efferosome 数据。 注意: 这允许使用单细胞方法进行数据分析, 从而允许对细胞间的变化进行量化。通过在单个实验中获得的单细胞数据, 可以对这些数据集进行人口级分析。Efferosome 特定的测量可以分析在人口水平, 单细胞水平和作为合奏 (如efferosomes 的人口独立于包含他们的细胞)15。</li>
	</ol></li>
</ol>6. 细胞凋亡细胞 Efferocytosis 检测方法
<ol><li>分别按照步骤1-3 中的描述制备 THP-1、J774.2 或 M2 巨噬细胞。如果需要, 细胞应该在进行任何实验之前, 用转基因或其他基因构造至少18小时进行转染。</li>
	<li>
在实验开始前的晚上, 按照4和5节中的说明准备凋亡 Jurkat 细胞, 并进行以下修改:
	<ol>
<li>稀释细胞并诱导细胞凋亡, 按 4.1–4.3, 收集所需数量的凋亡细胞为每5.3–5.4。</li>
		<li>添加一个细胞跟踪染料在制造商的建议浓度的凋亡细胞悬浮。在昏暗的室温下孵化20分钟的悬浮液。添加等量的 RPMI 1640 + 10% 的血清和孵化5分钟在室温下, 以淬火任何反应染料。不要为这些实验添加 NHS-生物素。</li>
		<li>颗粒细胞通过离心在 500 x g 5 分钟, 放弃上清, 并重新悬浮染色凋亡细胞100µL 的 RPMI 1640 + 10% 每井的巨噬细胞。</li>
	</ol>
</li>
	<li>如果需要, 从每一个井中移除培养基并添加500µL 的 PBS 来标记巨噬细胞, 其中包括制造商建议稀释与细胞追踪染料不重叠光谱与被添加到凋亡细胞或任何由巨噬细胞表达的荧光转基因。在室温下孵化20分钟, 然后在黑暗中加入等量的 DMEM + 10% 的血清, 在室温下孵育5分钟, 以淬火任何反应染料。</li>
	<li>把含巨噬细胞的盖玻片转移到莱顿室。添加400µL 的 DMEM + 10% 的血清, 其次是100µL 的标记凋亡细胞悬浮。通过轻轻吹打媒体2–3时间混合。</li>
	<li>将莱顿室转移到一个带电细胞荧光显微镜的加热和 CO2灌注腔内。 注: 对于缺乏 CO2灌注能力的显微镜设置, 使用用1毫米 4-(2 羟乙基)-1-piperazineethanesulfonic 酸 (HEPES) 缓冲的组织培养基, 而不是碳酸氢钠维持生理 pH 值, 而文化暴露在空气中。</li>
	<li>
捕获包含白光 (相位对比度或 DIC) 图像的时间间隔序列, 以及所有荧光标签的图像:
	<ol>
<li>使用100X 物镜的实验, 以解决细节是必要的 (如膜动力学吞噬凋亡细胞相互作用)。同时, 更一般的措施 (凋亡细胞摄取率, 巨噬细胞与凋亡的相互作用时间等) 最好用60X 或63X 的目标来成像。</li>
		<li>如果可用, 在单个捕获期间使用点访问来映像多个视图字段, 从而增加在单个实验中捕获的 efferocytic 事件的数量</li>
		<li>因为 efferocytes 经常吞食细胞凋亡的小片段, 而不是完整的细胞, 最好是捕捉 z 栈。为了最小化毒性, 捕获 z 段, 由显微镜目标的焦距 (通常是0.5–1.0 µm) 隔开, 通过细胞的厚度 (通常为8-10 µm 为巨噬细胞,例如8–20切片/细胞)。这将确保所有吞没和贩运事件在至少一个 z 平面上可见。或者, 使用大 (1–5µm 直径) 凋亡细胞模拟或凋亡细胞在 efferocytosis (如热休克中性粒细胞) 中发生极小的碎片, 而不是 z 堆积。我们已经描述了模拟和凋亡中性粒细胞以前28的制备。</li>
		<li>为了最小化漂白, 使用明亮的显影和捕捉图像, 利用捕获设置最小化漂白-例如低强度励磁结合高相机增益和短曝光时间29。我们强烈建议使用显微镜配备高灵敏度的电磁电荷耦合器件 (EM CCD) 相机或旋转盘共焦, 以及高速压电机械阶段, 为这种形式的成像。</li>
	</ol>
</li>
	<li>可以应用到这些时间推移视频的可能分析方法的数量是广泛的, 超出了我们在这里复习的能力。举例来说, 使用手动或自动跟踪软件跟踪15细胞内 efferosomes 的融合、裂变和运动, 用 endolysosomes 的定位分析来量化 efferosome 融合动力学间隔特定的荧光转基因13, 监测吸收过程, 如探测和杯形成30, 确定的招聘动态的信号和贩运蛋白质的 efferosome13, 和/或使用标有 pH 敏感、氧化剂敏感或蛋白酶活化显影31的凋亡细胞, 量化 efferosomes 的降解活性。</li>
</ol>

作者声明没有利益冲突。

本协议中概述的方法使动态 efferocytic 过程的成像和量化能够同时使用固定单元和活细胞方法。这些方法比一般使用的流式细胞术23,24提供了一些优势。使用固定样本进行内出染色, 可以更可靠、准确地量化 efferocytosis 的速率和程度, 事实上, 许多流式细胞术的方法只是标记凋亡细胞和不同显影的巨噬细胞, 并分数 efferocytosis 作为巨噬细胞与凋亡细胞核染色...

细胞凋亡, 或程序性细胞死亡, 是一种高度调控的生理过程, 发生在大多数多细胞有机体, 是关键的发展和稳态1。细胞凋亡除了参与正常细胞更替和胚胎发育外, 还能消除组织中受感染或受损的细胞, 并可因感染、炎症、癌症以及医疗干预而触发。如放疗或类固醇1。凋亡细胞在细胞表面暴露出 "吃-我" 信号, 在一系列专业和非专业吞噬中被受体识别, 统称为 "efferocytes"。这些受体的参与通过一个被称为 efferocytosis2,3的过程诱导 efferocyte 凋亡细胞的吸收和降解。磷脂是最具特色的食我信号驱动 efferocytosis。它通常局限于血浆膜的内小叶, 凋亡激活一个脂质 scramblase, 扰乱这种膜不对称, 从而暴露磷脂在细胞表面4。磷脂是在一些非凋亡细胞的细胞外表面发现的, 如成熟巨细胞和活化血小板。然而, 这些细胞不是 efferocytosed 由于存在 "不吃我" 信号, 例如 CD47, 在他们的细胞表面5,6,7。暴露的磷脂是由 efferocytes 表达的 efferocytic 受体阵列识别出来的。这些受体与磷脂的结合, 直接或通过 opsonins 的帮助, 激活信号通路, 促进吞没凋亡细胞成膜界的液泡称为 efferosome8,9,10,11,12. efferosome 与内涵体和溶酶体依次融合, 提供酸化 efferosome 所需的分子机械, 并降解凋亡细胞13、14。一旦降解, 凋亡细胞衍生材料被贩运到回收 endosome-一个过程, 限制免疫反应的凋亡细胞衍生抗原, 并可能允许从凋亡细胞13恢复养分, 15。efferocytosis 的失败导致凋亡细胞的清除受损;这些未清除细胞最终会经历次生坏死。坏死细胞释放亲炎症胞浆的内容, 病原体和自身抗原进入细胞外环境, 从而驱动一系列感染, 炎症和自身免疫性疾病16,17。同时, 细胞凋亡和 efferocytosis 促进了死亡和死亡细胞的清除, 并允许维持组织的稳态。
研究 efferocytosis 的分子机制需要方法, 提供一个明确的定量的凋亡细胞摄取。这种量化是复杂的事实, 不像其他吸收机制, 如吞和吞噬功能18,19, efferocytosis 可能不会导致吞没完整的目标细胞, 导致零碎吸收凋亡细胞的 efferocyte20。此处描述的协议描述了一种体外efferocytosis 分析, 它提供了对个体凋亡细胞内化和非内隐部分的精确划分, 并可与各种固定细胞和活细胞显微镜的方法。传统的吞噬功能检测在实验结束时添加特定于吞噬目标的抗体, 以标记非内化目标, 因为我们的方法不同于用共价键链接的生物素21标记凋亡目标。,22. 虽然在本试验中可以使用凋亡细胞特异抗体, 但 biotinylation 方法允许任何含蛋白质的靶标记, 并避免在执行染色时具有继发抗体交叉反应性的潜在问题.具体地说, 我们概述了细胞追踪染料和生物素双重染色的凋亡 Jurkat 细胞的制备。细胞追踪染料允许在 efferocytosis 期间追踪凋亡的细胞源材料, 而表面 biotinylation 则允许从 efferocytosed 凋亡细胞的非内化部分中内隐化。我们还描述了 J774.2 和 THP-1 细胞系的培养和制备, 用作小鼠和人的 efferocytes, 单核细胞衍生的 M2 巨噬细胞作为原电池 efferocytosis 的例子, Jurkat 细胞用作 efferocytic 靶。这些方法可以很容易地应用到其他细胞系或主细胞, 目标细胞发生任何形式的细胞死亡 (如凋亡, 坏死和 necroptosis), 以及微米大小模仿, 通过脂质涂层模拟凋亡细胞或efferocytic 受体所特有的配体涂层。
本协议中概述的方法比基于流式细胞术的方法在23、24领域中的应用有若干优点。通过对吞噬凋亡细胞相互作用的直接成像, 结合对全内和非内化凋亡细胞材料的清晰标记, 可进行 efferocytosis 定量测量。此外, 使用 pH 不敏感的显影限制混淆因素, 如抑制 FITC 和 GFP 荧光在溶酶酶的 pH 值混淆一些替代方法25。最后, 虽然没有详细描述, 这些方法可以使用 efferocytes 表达荧光标记转基因, 或与后固定染色, 以允许量化的信号分子活动和监测efferocytosis 过程中的细胞过程。

<table><tbody><tr><td>RPMI 1640 Media</td><td>Wisent</td><td>3500-000-EL</td><td></td></tr><tr><td>DMEM Media</td><td>Wisent</td><td>319-005-CL</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Wisent</td><td>080-150</td><td></td></tr><tr><td>PBS</td><td>Wisent</td><td>311-010-CL</td><td></td></tr><tr><td>18 mm circular glass coverslips #1.5 thickness</td><td>Electron Microscopy Sciences</td><td>72290-08</td><td>Size and shape of coverslip is not critical, but 18 mm fit into the wells of a standard 12-well plate which simplifies cell culture</td></tr><tr><td>Staurosporine</td><td>Cayman Chemical</td><td>81590</td><td>Dissolve in DMSO at 1 mM (1,000x stock solution)</td></tr><tr><td>Annexin V-Alexa 488</td><td>ThermoFisher</td><td>R37174</td><td></td></tr><tr><td>EZ-Link NHS-Biotin</td><td>ThermoFisher</td><td>20217</td><td>Store in a dessicator. Do not prepare a stock solution.</td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D2650</td><td></td></tr><tr><td>CellTrace FarRed</td><td>ThermoFisher</td><td>C34572</td><td></td></tr><tr><td>CellTrace Orange</td><td>ThermoFisher</td><td>C34851</td><td></td></tr><tr><td>Hoescht 33342</td><td>ThermoFisher</td><td>62249</td><td></td></tr><tr><td>FITC-Streptavadin</td><td>ThermoFisher</td><td>SA1001</td><td></td></tr><tr><td>Lympholyte-poly cell sepration medium</td><td>Cedarlane Labs</td><td>CL5071</td><td></td></tr><tr><td>Recombinant Human M-CSF</td><td>Peprotech</td><td>200-04</td><td></td></tr><tr><td>Recombinant Human IL-4</td><td>Peprotech</td><td>300-25</td><td></td></tr><tr><td>J774.2 Macrophage Cell Line</td><td>Sigma-Aldrich</td><td>85011428-1VL</td><td></td></tr><tr><td>THP-1 Human Monocyte Cell Line</td><td>ATCC</td><td>TIB-202</td><td></td></tr><tr><td>Jurkat T Cell Line</td><td>ATCC</td><td>TIB-152</td><td></td></tr></tbody></table>

quantification of efferocytosis by single-cell fluorescence microscopy

研究 efferocytosis 的调控需要能够准确量化凋亡细胞摄取的方法, 并探讨控制 efferocytosis 的信号和细胞过程。这种量化可能很难执行, 因为凋亡细胞往往 efferocytosed 零碎, 从而有必要的方法, 可以准确地划定的 efferocytosed 部分的凋亡目标与残留 unengulfed 细胞碎片。本文概述的方法利用双标记方法准确量化 efferocytes 的 efferocytosis 和 efferocytic 能力的动态, 如巨噬细胞。凋亡细胞的细胞质用细胞追踪染料进行标记, 以便对所有凋亡细胞源材料进行监测, 而凋亡细胞的表面 biotinylation 则允许在内化和非内内在之间进行分化。凋亡细胞分数。efferocytes 的 efferocytic 容量是通过对活体或固定细胞的荧光图像进行测定的, 并通过链亲和素染色的区分来定量测定束缚与内化目标的数量。这种方法比流式细胞术等方法具有多种优点, 即非 efferocytosed 与 efferocytosed 凋亡细胞分数的精确划分, 活细胞显微镜测量 efferocytic 动力学的能力, 以及在表达荧光标记转基因的细胞中对细胞信号进行研究的能力。结合, 本协议中概述的方法作为一个灵活的实验方法的基础, 可用于准确量化 efferocytic 活动和审问细胞信号通路活跃在 efferocytosis。

夜间培养的 Jurkat 细胞与1µM staurosporine 导致细胞凋亡 > 95%, 可证实与蛋白 V 染色 (图 1)。其他细胞类型可以用于这些实验, 虽然 staurosporine 的浓度和 staurosporine 治疗的时间将需要为每个细胞线进行优化。为了可靠的检测和定量的 efferocytosis, > 80% 的细胞应该是凋亡之前, 添加到 efferocytes。其他诱导细胞凋亡 (如热休克, 甙和紫外光) 也可以使用, 但?...

Watch this Scientific Journal Video about Quantification of Efferocytosis by Single-cell Fluorescence Microscopy at JoVE.com

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Efferocytosis, 凋亡细胞的吞噬去除, 是需要保持动态平衡, 并由受体和信号通路, 以允许识别, 吞没和内化的凋亡细胞的促进。在此, 我们提出了一个荧光显微镜协议的量化 efferocytosis 和 efferocytic 信号通路的活动。

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

quantification-of-efferocytosis-by-single-cell-fluorescence-microscopy

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.4K 次观看.  University of Western Ontario. Efferocytosis, 凋亡细胞的吞噬去除, 是需要保持动态平衡, 并由受体和信号通路, 以允许识别, 吞没和内化的凋亡细胞的促进。在此, 我们提出了一个荧光显微镜协议的量化 efferocytosis 和 efferocytic 信号通路的活动。

视频: Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular processes. This technique can be used on a variety of cell types, both adherent and non-adherent, to examine a variety of cellular properties. When studying phagocytosis, fluorometric technique utilizes phagocytic cell types such as macrophages, and fluorescently labeled opsonized particles whose fluorescence can be extinguished in the presence of trypan blue. Following plating of adherent macrophages in 96-well plates, fluorescent particles (green or red) are administered and cells are allowed to phagocytose for varied amounts of time. Following internalization of fluorescent particles, cells are washed with trypan blue, which facilitates extinction of fluorescent signal from bacteria which are not internalized, or are merely adhering to the cell surface. Following the trypan wash, cells are washed with PBS, fixed, and stained with DAPI (nuclear blue fluorescent label), which serves to label nuclei of cells. By a simple fluorometric quantification through plate reading of nuclear (blue) or particle (red/green) fluorescence we can examine the ratio of relative fluorescence units of green:blue and determine a phagocytic index indicative of amount of fluorescent bacteria internalized per cell. The duration of assay using a 96-well method and multichannel pipettes for washing, from end of phagocytosis to end of data acquisition, is less than 45 min. Flow cytometry could be used in a similar manner but the advantage of fluorometry is its high throughput, rapid method of assessment with minimal manipulation of samples and quick quantification of fluorescent intensity per cell. Similar strategies can be applied to non adherent cells, live labeled bacteria, actin polymerization, and essentially any process utilizing fluorescence. Therefore, fluorometry is a promising method for its low cost, high throughput capabilities in the study of cellular processes.

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

科研

免疫与感染

Preparation of Bead-supported Lipid Bilayers to Study the Particulate Output of T Cell Immune Synapses

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).

Here we present the protocol for the stepwise reconstitution of synthetic antigen-presenting cells using Bead-Supported Lipid Bilayers and their use to interrogate the synaptic output from activated T cells.

制备珠状支持脂质双分子层研究T细胞免疫突触的颗粒输出

Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) ...

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

Internalization and Observation of Fluorescent Biomolecules in Living Microorganisms via Electroporation

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing molecules of interest to fluorescent proteins such as GFP to study their expression, localization and function. However, GFP and its derivatives are significantly larger and less photostable than organic fluorophores generally used for in vitro experiments, and this can limit the scope of investigation. 
We recently introduced a straightforward, versatile and high-throughput method based on electroporation, allowing the internalization of biomolecules labeled with organic fluorophores into living microorganisms. Here we describe how to use electroporation to internalize labeled DNA fragments or proteins into Escherichia coli and Saccharomyces cerevisi&#230;, how to quantify the number of internalized molecules using fluorescence microscopy, and how to quantify the viability of electroporated cells. Data can be acquired at the single-cell or single-molecule level using fluorescence or FRET. The possibility of internalizing non-labeled molecules that trigger a physiological observable response in vivo is also presented. Finally, strategies of optimization of the protocol for specific biological systems are discussed.

Studies of biomolecules in vivo are crucial for understanding molecular function in a biological context. Here we describe a novel method allowing the internalization of fluorescent biomolecules, such as DNA or proteins, into living microorganisms. Analysis of in vivo data recorded by fluorescence microscopy is also presented and discussed.

通过电穿孔内在和生物分子荧光观察生活中的微生物

The ability to study biomolecules in vivo is crucial for understanding their function in a biological context. One powerful approach involves fusing ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

基于流式细胞术的突触材料小胶质细胞吞噬检测

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

作者聚焦：通过心理神经免疫学的技术创新促进理解

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

使用流式细胞术定量突触材料的小胶质细胞吞噬

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

神经科学

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular communication in various research fields; cancer biology, neuroscience, and drug delivery. Many EV uptake assays have been reported in the literature; however, there is a lack of practical, detailed experimental methodology. EV uptake can be assessed by fluorescently labeling EVs to detect their location within cells. Distinguishing between internalized EVs in cells and the superficial EVs on cells is difficult, yet critical, to accurately determine the EV uptake. Therefore, an assay that efficiently quantifies EV uptake through three-dimensional (3D) fluorescence confocal microscopy is proposed in this work. Fluorescently labeled EVs were prepared using a nano-filtration-based microfluidic device, visualized by 3D confocal microscopy, and then analyzed through advanced image-processing software. The protocol provides a robust methodology for analyzing EVs on a cellular level and a practical approach for efficient analysis.

Extracellular Vesicle Uptake Assay via Confocal Microscope Imaging Analysis

Extracellular vesicles (EVs) contribute to cellular biology and intercellular communications. There is a need for practical assays to visualize and quantify EVs uptake by the cells. The current protocol proposes the EV uptake assay by utilizing three-dimensional fluorescence imaging via confocal microscopy, following EV isolation by a nano-filtration-based microfluidic device.

通过共聚焦显微镜成像分析进行细胞外囊泡摄取测定

There is a need for practical assays to visualize and quantify the cells' extracellular vesicle (EV) uptake. EV uptake plays a role in intercellular ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

通过纳流式细胞术表征单个细胞外囊泡跨膜蛋白

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

单细胞荧光显微镜定量 Efferocytosis 的研究

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