单细胞荧光显微镜定量 Efferocytosis 的研究
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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)。 每天通过轻轻摇动烧瓶, 将细胞均匀地…
Representative Results
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?…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| RPMI 1640 Media | Wisent | 3500-000-EL | |
| DMEM Media | Wisent | 319-005-CL | |
| Fetal Bovine Serum (FBS) | Wisent | 080-150 | |
| PBS | Wisent | 311-010-CL | |
| 18 mm circular glass coverslips #1.5 thickness | Electron Microscopy Sciences | 72290-08 | 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 |
| Staurosporine | Cayman Chemical | 81590 | Dissolve in DMSO at 1 mM (1,000x stock solution) |
| Annexin V-Alexa 488 | ThermoFisher | R37174 | |
| EZ-Link NHS-Biotin | ThermoFisher | 20217 | Store in a dessicator. Do not prepare a stock solution. |
| DMSO | Sigma-Aldrich | D2650 | |
| CellTrace FarRed | ThermoFisher | C34572 | |
| CellTrace Orange | ThermoFisher | C34851 | |
| Hoescht 33342 | ThermoFisher | 62249 | |
| FITC-Streptavadin | ThermoFisher | SA1001 | |
| Lympholyte-poly cell sepration medium | Cedarlane Labs | CL5071 | |
| Recombinant Human M-CSF | Peprotech | 200-04 | |
| Recombinant Human IL-4 | Peprotech | 300-25 | |
| J774.2 Macrophage Cell Line | Sigma-Aldrich | 85011428-1VL | |
| THP-1 Human Monocyte Cell Line | ATCC | TIB-202 | |
| Jurkat T Cell Line | ATCC | TIB-152 |
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Citazione di questo articolo
Taruc, K., Yin, C., Wootton, D. G., Heit, B. Quantification of Efferocytosis by Single-cell Fluorescence Microscopy. J. Vis. Exp. (138), e58149, doi:10.3791/58149 (2018).