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的分数, 因而缺乏区分界限与内化凋亡细胞材料的能力。替代流式细胞术方法包括那些使用 pHrodo 标记凋亡细胞24。pHrodo 是 ph 敏感的荧光, 在酸性 ph 值的亮度增加。虽然这荧光提供了更好的解决之间的非内化和内化的材料, 因为荧光增加具体后, 凋亡细胞的内化为和酸化的 efferosome, 结果可以混淆的疾病过程, 损害 efferosome 酸化34,35, 这一方法将错过 efferosomes 早期 (前酸化) 阶段的 efferocytosis36, 位于酸化贫困地区的细胞27, 或在弱酸化其溶酶体37的细胞中。本协议中描述的方法的第二个优点是使用活细胞成像测量 efferocytosis 的动力学, 如探测、efferocytic 突触的形成以及 efferosomes 细胞内的贩运不能使用基于流式细胞仪的方法检测到。
虽然这种方法提供了许多优点, 但需要对许多数以百计的细胞进行量化的实验,例如检测罕见事件的实验, 或量化高度可变的过程--可能很难, 分析这些大型数据集花费了过多的时间。高通量成像方法 (如成像流式细胞术38 ) 可能比常规显微镜具有更高的吞吐量, 尽管在我们的经验中, 当前的自动化图像分析程序并不总是能够准确地分割非内部化与内化材料。具体地说, 在凋亡细胞和 efferocyte 之间形成的紧密的 efferocytic 突触可以排除链亲和素染色, 因此在细胞追踪染料通道中, 这种有界凋亡细胞经常出现为固体质量, 部分被链亲和素染色 (图 2)。因此, 虽然 efferosomes 是容易识别的算法, 凋亡细胞的绑定部分往往认错作为 efferosome 由于难以核算的部分链亲和素染色。虽然我们还没有找到一个程序, 可以准确地识别和量化的碎片摄取细胞凋亡没有人类的帮助, 我们发现, 可训练半自动系统39可以大大加快分析, 减少分数efferocytosed 测量从2–3分钟到 < 六十年代每细胞。另外, 可以使用非易消化的凋亡细胞模拟或细胞类型, 这是最小的片段在细胞凋亡。这简化了对单个、更大结构的检测, 这可能更适于自动化方法, 并且可能会消除对 z 堆叠的需要。即使有了这些进展, 这种方法的获取和分析速度仍然是有限的, 当需要对大细胞数进行分析时, 这种流式细胞术仍然是最可行的方法23。
虽然我们已经描述了这种方法, 以细胞线和原发性人巨噬细胞作为 efferocytes 和凋亡 Jurkat 单元 (T 细胞线) 作为目标, 这种方法可以应用于任何 efferocytic 细胞类型或凋亡细胞目标。事实上, 类似的方法已经被用来调查 efferocytosis 肝细胞和上皮细胞9,40,41, 和 efferocytosis 的临床相关靶点, 如肿瘤细胞42。在使用非免疫 efferocytes 或建模特定的 efferocytic 事件时, 可能需要修改此协议。例如, Jurkat 细胞是不粘附的, 因此不太可能完全重述在与粘附凋亡细胞或凋亡细胞在固体组织中相互作用 efferocytes 遇到的机械力和空间限制。许多细胞类型在细胞凋亡过程中会保持黏附力, 因此可以作为黏附靶;作为一个例子, HeLa 细胞重现性接受 staurosporine 诱导的凋亡, 磷脂扰, 起泡在第4-6 h 期, 同时保持细胞体的黏附力43。在胶原基质或干细胞衍生的 organoids44中悬浮的细胞可能是研究固体组织 efferocytosis 的潜在模型, 尽管我们不知道任何使用这些方法的研究。对于一些模型, 免疫细胞, 如 Jurkat 细胞和中性粒细胞不应该被用作凋亡靶, 因为这些细胞可以释放基于细胞因子的 "发现-我" 信号, 如 CX3CL1, 这可能是一个混杂的因素在模型中, 炎症或迁移过程被调查45。因此, 虽然这种检测的通用性允许它被用来探索跨一系列细胞类型和模型系统的 efferocytosis, 必须注意选择适当的 efferocytes, 靶细胞和培养条件, 以最佳模式的生理正在调查中的过程。
本协议中描述的分析方法仅作为一个起点, 这些实验的基于成像的性质能够进行范围广泛的分析。例如, efferosome 定位和直径测量可以收集时, 执行分数 efferocytosed 测量, 或测量在活细胞时间-课程, 以调查过程, 如细胞内贩运efferosomes 和凋亡细胞降解率13,15。染色 (图 4) 可以用来调查蛋白质的招募 efferosomes, 而活细胞成像结合形态学分析可以用来量化的过程, 这种约束力的功效和率的吞没30,46. 我们经常在表达荧光转基因的巨噬细胞中进行这些化验, 报告信号分子活化或 efferocytosis 期间细胞事件活动 (图 6)。这些记者能够在 efferocytosis 期间对信号过程进行监测;例如, 我们使用荧光标记的 GTPases 来探讨 Rab5、Rab7 和 Rab17 在调解 efferosome 处理和随后贩运凋亡细胞衍生抗原13,15中的作用。同样, 将细胞死亡、efferosome pH 值、活性氧种类生产以及其他细胞过程纳入活细胞实验的记者可以用来调查 efferosomes 31 的降解过程. ,47。在这些实验中, 一个共同的挑战是识别转基因或记者与兼容的显影;通常, 我们将消除细胞跟踪染料和/或链亲和素的自由通道, 以成像其他显影。对于一些实验来说, 用非分割模拟代替凋亡细胞是有益的。这允许定量的信号动力学和细胞过程, 而不复杂的跟踪从一个单一的凋亡细胞衍生的多 efferosomes。见埃文斯等人有关制备凋亡细胞模拟28的说明。
在设计这些实验时最常见的困难是找到显影的组合, 使所需的过程能够被成像, 同时最小化漂白和毒性29。荧光的选择主要由激光/励磁过滤器、dichroics/立方体和显微镜的发射过滤器决定, 因此显影的选择往往是个别显微镜所特有的。通常, 较长的波长显影 (橙色到远红色,例如发射极大值 > 580 nm) 不太容易漂白, 这些波长对细胞的破坏性较小。绿色显影 (发射极大值 ~ 525 nm) 可以使用, 但需要注意的是限制毒性细胞。显影, 激发在波长少于480毫微米 (紫罗兰和蓝色) 应该避免由于他们的高毒性和倾向为这些励磁波长漂白其他显影29。在可能的情况下, 应选择高亮度和稳定的显影。同样, 应调整图像采集参数, 以最小化漂白和毒性-例如, 在低励磁强度下较长的曝光比较高的励磁强度高48。添加抗氧化剂, 如芦丁和去除一些培养基成分可以改善钴和减少毒性49。即使仔细选择显影和成像条件, 限制漂白的需要往往需要捕获低信噪比的图像 (如图 5所示)。如果量化荧光强度, 需要很大的注意, 以限制图像处理文物;理想情况下, 不需要对未加工任何形式的原始图像进行分析。如果 deconvolving 图像在量化之前, 一个保留荧光强度的反褶积算法必须使用50,51。
活细胞收购可能特别具有挑战性, 成功的实验需要在激发强度、曝光时间、帧速率和实验持续时间之间进行仔细的平衡。激发强度和曝光时间应调整, 以尽量减少毒性如上所述, 警告说, 较长的曝光时间可能导致运动文物由于细胞移动或限制的帧率的收购。帧速率可以根据实验要求而变化。帧率较低 (帧之间的5–30最小) 允许在长时间内 (12 小时或更多) 进行成像, 但提供了有关诸如膜动力学和 efferosomes 后 efferocytosis 贩运等现象的最低数据。高帧率 (每秒1帧) 提供了很好的时间分辨率的 efferocytic 事件和 efferosome 贩运-但即使谨慎选择显影, 激发强度和曝光时间-漂白或毒性通常将这些实验限制在不到一个小时的时间内。根据我们的经验, 在 2–6 h 的实验期内, 获取图像每一分钟都是一个可以接受的折衷, 提供足够数量的 efferocytic 事件的量化图像, 并具有合理的时间分辨率。
改变和失败的 efferocytosis 被称为参与癌症, 动脉粥样硬化和多种自身免疫性疾病的病理学, 与 efferocytosis 靶向治疗显示伟大的临床承诺7,52, 53,54,55。在这些领域的进一步发展将需要识别和鉴定细胞过程, 受体和信号通路, 调节 efferocytosis。本议定书中提出的分析是这些研究的有力工具, 可以修改以量化许多调节 efferocytosis 的细胞和信号过程。

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, 但根据我们的经验, 这些产生更多的异种诱导细胞凋亡, 并导致混合细胞群体中含有凋亡, 继发坏死和非凋亡靶细胞。
对于内出染色的固定细胞成像, 应在所有时间点密切并列 efferocyte 凋亡细胞相互作用, 明确划定非 efferocytosed (链亲和素+/细胞跟踪染料+) 和efferocytosed (链亲和素/细胞追踪+) 材料可见 (图 2)。重要的是要注意的是, 在 efferocyte 和凋亡细胞之间形成的突触, 往往足够紧密, 以排除链亲和素, 因此任何细胞跟踪染料染色对象轴承链亲和素在其圆周上的任何点应视为绑定单元格而不是 efferosome。在大多数实验中, 被 efferocytosed 的凋亡细胞的分数会随着时间的推移而增加, 直到没有非内化的凋亡细胞材料残留, 或者直到吞噬达到最大的 efferocytic 容量 (图3). 分析通常是针对单个单元进行和记录的 (图 3A), 但是, 数据可以在单个实验中的单元格中平均分布, 并且通过常规方法分析多个实验重复值的平均值。统计方法 (图 3B)。修改这一标准化的协议可以使使用的主要细胞类型, 替代 efferocytic 目标, 并为其他染色将包括在内。例如,图 4显示了一个主要的 M2-polarized 人巨噬细胞, efferocytosed 凋亡细胞模拟由3µm 直径的二氧化硅珠涂在磷脂和卵磷脂28的混合物中, 其次是随后的固定, 通透和染色的回收 endosome 标记 Rab17。
在活细胞成像 efferocytic 专业吞噬将被观察到扩展和收回小进程的过程中称为 "探测"30 (图 5, t = 0);当这些过程遇到目标时, 它们会坚定地坚持目标并将其绘制到吞噬。这种探测活动可能没有观察到非专业吞噬, 如上皮细胞。然后 efferocyte 将形成一个紧密的 "efferocytic 突触"32在本身和凋亡细胞之间, 这种突触经常包裹凋亡细胞的一个很大一部分 (图 5, t = 10)。然后, efferocyte 将从这个突触中提取凋亡细胞的碎片到其细胞质 (图 5, 10–30分钟)。在他们的形成之后, 这些新生的 efferosomes 被贩卖了从突触和对围核区域, 起因于 Rab7/RILP/dynein-dynactin 斡旋的运输33。随着时间的推移, efferosomes 将萎缩, 结果退化的材料重新分配到整个细胞, 在那里他们可能被吸收或回收13,15。检测这些过程的能力高度依赖于实验的采集帧速率和持续时间。快速的过程, 如探测可能不会被观察在较低帧率, 而慢事件 (efferosome 贩运和吸收) 需要更长的成像周期-在这两种情况下, 这些大型图像采集往往需要捕捉低强度图像限制漂白和毒性 (图 5)。与固定细胞检测一样, 这种检测的活细胞版本可以根据调查者的需要进行修改。图 6显示了 J774.2 巨噬细胞的活细胞采集单帧, 表达转基因荧光丹麦血浆膜 (PM-GFP), 并有选择地结合信号脂 PI (3) P (FYVE RFP)。efferosome 膜上的 PI (3) P 的生成可以被检测为 FYVE-GFP+ efferosome 膜的协同定位。通过量化 FYVE efferosome 的强度, 可以对 efferosome 上 PI (3) P 信号的动力学进行量化 (图 6)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58149/58149fig1.jpg"> 图 1: 蛋白 V 染色的凋亡和非凋亡 Jurkat 细胞.蛋白 V 标签的磷脂 "吃我" 信号暴露的凋亡细胞。顶未经治疗的 (UT) Jurkat 细胞显示健康 (平滑和圆形) 形态学 (DIC), 不染色与蛋白 v (底部) Jurkat 细胞夜间培养1µM staurosporine (STS) 采取凋亡形态学 (不规则和 blebbed) 和染色蛋白 v. 缩放条 = 10 µm, 图像强度显示为彩色地图。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58149/58149fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58149/58149fig2.jpg"> 图 2: J774.2 巨噬细胞凋亡的早期和晚期吞没.图像是巨噬细胞在早期 (60 分钟) 和晚 (120 分钟) 的 efferocytosis 阶段。白光 (DIC) 图像显示巨噬细胞 (mφ) 与凋亡细胞 (AC) 之间紧密的界面。细胞追踪染料 (温、红) 揭示所有凋亡细胞源材料的位置。FITC-链亲和素染色 (Str, 绿色) 识别凋亡细胞尚未被巨噬细胞吞噬的部分。巨噬细胞细胞核预染 DAPI (蓝色)。Efferosomes (红色) 与凋亡细胞 (绿色/黄色) 的非内化部分在链亲和素和细胞追踪染料图像的叠加中容易辨认。注意在早期 timepoint 的巨噬细胞凋亡细胞膜上没有链亲和素染色, 这是由被严密形成的 efferocytic 突触 (*) 排除链亲和素所造成的。巨噬细胞核由赫斯特染色 (蓝色) 鉴定。刻度条 = 10 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58149/58149fig2large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58149/58149fig3.jpg"> 图 3: J774.2 巨噬细胞 Efferocytosed 凋亡淋巴细胞分数的时间过程.采用细胞追踪染料/Streptavadin 染色法, 对所述时间进行 Efferocytosis 测定, 并在各时间点确定吞噬凋亡细胞材料的分数。(A) 在单个巨噬细胞内 efferocytosed 材料的分数, 数据是从各个单元显示出来的, 水平条表示平均值。12–17细胞每条件, 数据从 1 5 独立实验。(B) efferocytosed 材料的分数, 平均5个独立实验, 至少10个细胞/条件/重复。* p < 0.05 相比, 30 分钟, 克鲁斯卡尔-沃利斯测试与邓恩矫正。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58149/58149fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58149/58149fig4.jpg"> 图 4: Rab17 对 Efferosomes 在原发性人巨噬细胞中的招募分析.M2-polarized 巨噬细胞吞噬凋亡细胞模拟 (箭) 和 immunostained Rab17 (绿色) 60 分钟后吞没。鳞片棒 = 5 µm 细胞核染色 Hoeschst, DIC 图像显示细胞形态学, 并用于识别凋亡细胞模拟。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58149/58149fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58149/58149fig5.jpg"> 图 5: 活细胞 efferocytosis 测定.细胞跟踪染料 labeledJ774.2 巨噬细胞 (mφ, 绿色) 被记录, 因为它吞噬了一个凋亡 Jurkat 细胞标记与不同的彩色细胞跟踪染料 (AC, 红色)。凋亡细胞在内化过程中被分解成多个片段, 导致多 efferosomes (箭) 的逐段吸收和形成。刻度条 = 10 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58149/58149fig5large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58149/58149fig6.jpg"> 图 6: 使用荧光转基因研究 Efferocytic 信号.J774.2 巨噬细胞以 GFP 标记的血浆膜标记物 (PM、绿色) 和 FYVE-RFP 结构转染, 结合信号脂 PI (3) P (红色)。Efferocytosis 形成一个新生的血浆膜衍生的 efferosome, 其中含有 PI (3) P, 由 FYVE-RFP 招募 (箭头) 检测到。PI (3) P 信令的动力学被量化为相对于 efferosome 闭合点 (t = 0, 图) 上的 FYVE RFP 信号的褶皱强度。本实验采用凋亡细胞模拟珠 (箭头, DIC) 而不是凋亡细胞。刻度条 = 5 µm.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58149/58149fig6large.jpg" target="_blank">请点击这里查看这个数字的大版本.</a>

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

Nanyang Technological University

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

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

科研

免疫与感染

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

单细胞分析枯草芽孢杆菌生物膜，用荧光显微镜和流式细胞仪

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

细菌毒素引起的响应，用活细胞荧光显微镜的可视化

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

Single Cell Measurements of Vacuolar Rupture Caused by Intracellular Pathogens

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed compartment allows bacteria to move within the cytosol, proliferate and further invade neighboring cells. Mycobacterium tuberculosis is phagocytosed by immune cells, and has recently been shown to rupture phagosomal membrane in macrophages. We developed a robust assay for tracking phagosomal membrane disruption after host cell entry of Shigella flexneri or Mycobacterium tuberculosis. The approach makes use of CCF4, a FRET reporter sensitive to &beta;-lactamase that equilibrates in the cytosol of host cells. Upon invasion of host cells by bacterial pathogens, the probe remains intact as long as the bacteria reside in membrane-enclosed compartments. After disruption of the vacuole, &beta;-lactamase activity on the surface of the intracellular pathogen cleaves CCF4 instantly leading to a loss of FRET signal and switching its emission spectrum. This robust ratiometric assay yields accurate information about the timing of vacuolar rupture induced by the invading bacteria, and it can be coupled to automated microscopy and image processing by specialized algorithms for the detection of the emission signals of the FRET donor and acceptor. Further, it allows investigating the dynamics of vacuolar disruption elicited by intracellular bacteria in real time in single cells. Finally, it is perfectly suited for high-throughput analysis with a spatio-temporal resolution exceeding previous methods. Here, we provide the experimental details of exemplary protocols for the CCF4 vacuolar rupture assay on HeLa cells and THP-1 macrophages for time-lapse experiments or end points experiments using Shigella flexneri as well as multiple mycobacterial strains such as Mycobacterium marinum, Mycobacterium bovis, and Mycobacterium tuberculosis.

We describe a method for tracking the endomembrane rupture elicited by the intracellular bacteria Shigella flexneri and Mycobacterium tuberculosis upon host cell invasion. Our assay makes use of CCF4, a host cytoplasmic FRET probe in live or fixed cells. This reporter is degraded by an enzyme activity present on the bacterial surface.

单细胞测量液泡破裂引起细胞内病原体

Shigella flexneri are pathogenic bacteria that invade host cells entering into an endocytic vacuole. Subsequently, the rupture of this membrane-enclosed ...

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4&#39;,6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells.

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells.

荧光显微镜方法测定细菌在协会的可行性与哺乳动物细胞

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols ...

Assessing Anti-fungal Activity of Isolated Alveolar Macrophages by Confocal Microscopy

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic cells that encounter inhaled fungi and other microbes. Macrophages and other immune cells recognize Aspergillus motifs by pathogen recognition receptors and initiate downstream inflammatory responses. The phagocyte NADPH oxidase generates reactive oxygen intermediates (ROIs) and is critical for host defense. Although NADPH oxidase is critical for neutrophil-mediated host defense1-3, the importance of NADPH oxidase in macrophages is not well defined. The goal of this study was to delineate the specific role of NADPH oxidase in macrophages in mediating host defense against A. fumigatus. We found that NADPH oxidase in alveolar macrophages controls the growth of phagocytosed A. fumigatus spores4. Here, we describe a method for assessing the ability of mouse alveolar macrophages (AMs) to control the growth of phagocytosed Aspergillus spores (conidia). Alveolar macrophages are stained in vivo and ten days later isolated from mice by bronchoalveolar lavage (BAL). Macrophages are plated onto glass coverslips, then seeded with green fluorescent protein (GFP)-expressing A. fumigatus spores. At specified times, cells are fixed and the number of intact macrophages with phagocytosed spores is assessed by confocal microscopy.

A method to evaluate the ability of isolated mouse alveolar macrophages to control the growth of phagocytosed Aspergillus spores by confocal microscopy.

评估隔离肺泡巨噬细胞的激光共聚焦显微镜抗真菌活性

The lung is an interface where host cells are routinely exposed to microbes and microbial products. Alveolar macrophages are the first-line phagocytic ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like Shigella flexneri and Francisella novicida need to induce the rupture of the phagosome. In contrast, Salmonella typhimurium replicates in a vacuolar compartment, known as Salmonella-containing vacuole (SCV). However certain mutants of Salmonella fail to maintain SCV integrity and are thus released into the cytosol. The percentage of cytosolic vs. vacuolar bacteria on the level of single bacteria can be measured by differential permeabilization, also known as phagosome-protection assay. The approach makes use of the property of detergent digitonin to selectively bind cholesterol. Since the plasma membrane contains more cholesterol than other cellular membranes, digitonin can be used to selectively permeabilize the plasma membrane while leaving intracellular membranes intact. In brief, following infection with the pathogen expressing a fluorescent marker protein (e.g. mCherry among others), the plasma membrane of host cells is permeabilized with a short incubation in digitonin containing buffer. Cells are then washed and incubated with a primary antibody (coupled to a fluorophore of choice) directed against the bacterium of choice (e.g. anti-Salmonella-FITC) and washed again. If unmarked bacteria are used, an additional step can be done, in which all membranes are permeabilized and all bacteria stained with a corresponding antibody. Following the staining, the percentage of vacuolar and cytosolic bacteria can be quantified by FACS or microscopy by counting single or double-positive events. Here we provide experimental details for use of this technique with the bacterium Salmonella typhimurium. The advantage of this assay is that, in contrast to other assay, it provides a quantification on the level of single bacteria, and if analyzed by microscopy provides the exact number of cytosolic and vacuolar bacteria in a given cell.

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

We describe the quantification of cytosolic and vacuolar Salmonella typhimurium in bone-marrow derived macrophages using differential digitonin permeabilization.

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Measuring Phagosome pH by Ratiometric Fluorescence Microscopy

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or neutralize the ingested material, or to present it as antigens and initiate adaptive immune responses. The pH of phagosomes is a critical parameter regulating fission or fusion with endomembranes and activation of proteolytic enzymes, events that allow the phagocytic vacuole to mature into a degradative organelle. In addition, translocation of H+ is required for the production of high levels of reactive oxygen species (ROS), which are essential for efficient killing and signaling to other host tissues. Many intracellular pathogens subvert phagocytic killing by limiting phagosomal acidification, highlighting the importance of pH in phagosome biology. Here we describe a ratiometric method for measuring phagosomal pH in neutrophils using fluorescein isothiocyanate (FITC)-labeled zymosan as phagocytic targets, and live-cell imaging. The assay is based on the fluorescence properties of FITC, which is quenched by acidic pH when excited at 490 nm but not when excited at 440 nm, allowing quantification of a pH-dependent ratio, rather than absolute fluorescence, of a single dye. A detailed protocol for performing in situ dye calibration and conversion of ratio to real pH values is also provided. Single-dye ratiometric methods are generally considered superior to single wavelength or dual-dye pseudo-ratiometric protocols, as they are less sensitive to perturbations such as bleaching, focus changes, laser variations, and uneven labeling, which distort the measured signal. This method can be easily modified to measure pH in other phagocytic cell types, and zymosan can be replaced by any other amine-containing particle, from inert beads to living microorganisms. Finally, this method can be adapted to make use of other fluorescent probes sensitive to different pH ranges or other phagosomal activities, making it a generalized protocol for the functional imaging of phagosomes.

Phagosomal pH influences phagosome maturation, oxidant production, phagosomal killing as well as antigen presentation. Here we describe a ratiometric method for measuring time-course and endpoint pH changes in individual phagosomes in living phagocytes using fluorescence microscopy.

由成比例的荧光显微镜测量吞噬体pH值

Phagocytosis is a fundamental process through which innate immune cells engulf bacteria, apoptotic cells or other foreign particles in order to kill or ...

A Simple Fluorescence Assay for Quantification of Canine Neutrophil Extracellular Trap Release

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although a component of the innate immune response, NETs are implicated in a range of disease processes including autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay. Blood is collected using conventional venipuncture techniques. Neutrophils are isolated using dextran sedimentation and a density gradient using conditions optimized for dog blood. After allowing time for attachment to the wells of a 96 well plate, neutrophils are treated with NET-inducing agonists such as phorbol-12-myristate-13-acetate or platelet activating factor. DNA release is measured by the fluorescence of a cell-impermeable nucleic acid dye. This assay is a simple, inexpensive method for quantifying NET release, but NET formation rather than other causes of cell death must be confirmed with alternative methods.

Neutrophil extracellular traps (NETs) are networks of DNA, histones and neutrophil proteins. Although a component of the innate immune response, NETs are implicated in autoimmunity and thrombosis. This protocol describes a simple method for canine neutrophil isolation and quantification of NETs using a microplate fluorescence assay.

一个简单的荧光试验犬中性粒细胞胞外陷阱里放的量化

Neutrophil extracellular traps are networks of DNA, histones and neutrophil proteins released in response to infectious and inflammatory stimuli. Although ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.