1] Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid, Spain. [2].
Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid, Spain.
Nat Protoc. 2014 May;9(5):1102-12. doi: 10.1038/nprot.2014.078. Epub 2014 Apr 17.
Although there are multiple methods for analyzing apoptosis in cultured cells, methodologies for analyzing apoptosis in vivo are sparse. In this protocol, we describe how to detect apoptosis of leukocytes in mouse lymph nodes (LNs) via the detection of apoptotic caspases. We have previously used this protocol to study factors that modulate dendritic cell (DC) survival in LNs; however, it can also be used to analyze other leukocytes that migrate to the LNs. DCs labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled DCs reach the popliteal LN (PLN), the animals are intravenously injected with FLIVO, a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted PLNs are then examined under a two-photon microscope to look for the presence of apoptotic cells among the DCs injected. The protocol requires 6-6.5 h for preparation and analysis plus an additional 34-40 h to allow apoptosis of the injected DCs in the PLN.
虽然有多种方法可用于分析培养细胞中的细胞凋亡，但体内分析细胞凋亡的方法却很少。在本方案中，我们将描述如何通过检测凋亡半胱氨酸蛋白酶来检测小鼠淋巴结（LN）中白细胞的凋亡。我们之前曾使用该方案研究调节 LN 中树突状细胞（DC）存活的因素；但是，它也可用于分析迁移到 LN 的其他白细胞。将荧光细胞示踪剂标记的 DC 经皮注射到小鼠后脚掌。一旦标记的 DC 到达腘淋巴结（PLN），则通过静脉内注射 FLIVO，一种可渗透的荧光试剂，该试剂可选择性标记活性半胱天冬酶，从而标记凋亡细胞。然后，在双光子显微镜下检查取出的 PLN，以寻找注射的 DC 中是否存在凋亡细胞。该方案的准备和分析需要 6-6.5 小时，另外还需要 34-40 小时以使注入 PLN 的 DC 凋亡。
