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Detecting apoptosis of leukocytes in mouse lymph nodes

Abstract

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.

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Figure 1: Cartoon showing the FLICA-based strategy used to detect apoptotic leukocytes.
Figure 2: Hoechst and FLIVO staining of apoptotic leukocytes.
Figure 3: Two-photon microscopy analysis of CMFDA-DCs showing SR-FLIVO staining in explanted LNs.
Figure 4: Effects of pharmacological agents on the survival of DCs in the PLN.

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Acknowledgements

We acknowledge the criticism of O. Criado and J. Torres Bacete. This study is supported by grants awarded by Ministerio de Educación y Ciencia (SAF2005-00801), Ministerio de Economía y Competitividad (SAF2011-23890), RIER (RETICS Program/Instituto de Salud Carlos III) (RD08/0075) and Consejería de Educación y Empleo from Comunidad de Madrid (Raphyme, S2010/BMD-2350). L.G.-C. was supported by a Formación de Profesorado Universitario (FPU) scholarship (Ministerio de Educación y Ciencia). P.L.-C. was supported by a Formación de Personal Investigador (FPI) scholarship (Ministerio de Economía y Competitividad). C.E.-D. was the recipient of an I3P fellowship (Consejo Superior de Investigaciones Científicas-Fondo Social Europeo). C.D.-M. was partially supported by an FPI fellowship conferred by the Ministerio de Educación y Ciencia (Spain) and by a contract associated with grant no. RD08/0075 (RIER). L.R.-B. was partially supported by a scholarship associated with grant no. PI021058.

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L.G.-C., C.D.-M., P.L.-C. and C.E.-D. performed the experiments. L.G.-C., C.D.-M., P.L.-C., C.E.-D., L.M.A.-C., L.R.-B. and J.L.R.-F. analyzed the data. J.L.R.-F. wrote the manuscript with input from all authors.

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Correspondence to José Luis Rodríguez-Fernández.

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Gómez-Cabañas, L., Delgado-Martín, C., López-Cotarelo, P. et al. Detecting apoptosis of leukocytes in mouse lymph nodes. Nat Protoc 9, 1102–1112 (2014). https://doi.org/10.1038/nprot.2014.078

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