Detecting apoptosis of leukocytes in mouse lymph nodes

Developers

Laura Gómez-Cabañas, Cristina Delgado-Martín, Lorena Riol-Blanco, José Luis Rodríguez-Fernández et al.

Description of the technology

Although there are multiple methods for analyzing apoptosis in cultured cells (e.g. transferase-mediated dUTP nick-end labeling (TUNEL), flow cytometry of cell suspensions for the presence of apoptotic markers on the membrane, etc), methodologies for analyzing apoptosis in vivo are sparse.

Main element of this technology is formulated as a protocol, describing how to detect apoptosis of leukocytes in mouse lymph nodes via the detection of apoptotic caspases. This protocol have previously used to study factors that modulate dendritic cell survival in lymph nodes; however, it can also be used to analyze other leukocytes that migrate to the lymph nodes. Dendritic cells labeled with a fluorescent cell tracker are subcutaneously injected in the posterior footpads of mice. Once the labeled dendritic cells reach the popliteal lymph nodes, the animals are intravenously injected with fluorescent dye-conjugated apoptotic detector (FLIVO − fluorescence in vivo), a permeant fluorescent reagent that selectively marks active caspases and consequently apoptotic cells. Explanted popliteal lymph nodes are then examined under a two-photon microscope to look for the presence of apoptotic cells among the dendritic cells injected. The protocol requires 6–6.5 h for preparation and analysis plus an additional 34–40 h to allow apoptosis of the injected dendritic cells in the popliteal lymph nodes.

Practical application

An important advantage protocol according to this technology is its applicability to study the survival of leucocytes and, especially, dendritic cells in vivo. This protocol can also be used to study the survival of other leukocytes that migrate to the lymph nodes. In this regard, the protocol can be used to study any cell type that expresses the chemokine receptor CCR7, which is responsible for driving leukocytes and other cells to the lymph nodes. The cell types that migrate to the lymph nodes include B cells, naive and central memory T cells and some types of monocytes and macrophages.

Notably, some metastatic cells, including cells of melanoma, non-small lung cancer, gastric cancer and esophageal cancer also express CCR7 and/or CXCR4 and can migrate to the lymph nodes, which implies that the survival of these cells or their apoptosis in response to different treatments can also be readily analyzed by using this strategy.

The method can also be used to analyze the survival of leukocytes or other cells types, not only in mice but also in other species, provided that the cells to be studied use caspase-mediated mechanisms of apoptosis, and that they can be tracked, either by labeling with intravital markers or by other alternative strategies for subsequent analysis by two-photon microscopy.

Thus, the technology can be promising for application in the study of different diseases (immune diseases, cancer, etc), processes of aging, including cell senescence, drug discovery and others.

Laboratories

• Centro de Investigaciones Biológicas. Consejo Superior de Investigaciones Científicas, Madrid (Spain)
• Centro de Microscopía y Citometría, Universidad Complutense, Madrid (Spain)
• Present address: Department of Microbiology and Immunobiology, Harvard Medical School, Boston (USA)

Links

Publications

• Gómez-Cabañas, L. et al. «Detecting apoptosis of leukocytes in mouse lymph nodes." 9 Nature Protocols (2014): 1102–1112.
• Delgado-Martin, C. et al. «Chemokine CXCL12 uses CXCR4 and a signaling core formed by bifunctional Akt, extracellular signal-regulated kinase (ERK)½, and mammalian target of rapamycin complex 1 (mTORC1) proteins to control chemotaxis and survival simultaneously in mature dendritic cells." 286 J. Biol. Chem. (2011): 37222–37236.
• Rey-Gallardo, A. et al. «Polysialic acid is required for neuropilin-2a/b-mediated control of CCL21-driven chemotaxis of mature dendritic cells and for their migration in vivo." 21 Glycobiology (2011): 655–662.