Authors
Cristina Delgado-Martn, Lorena Riol-Blanco, Luis M Alonso-C, and Jos Luis Rodrguez-Fernndez
Summary
This protocol describes a method to detect and measure the percentage of apoptotic or live dendritic cells (DCs) in popliteal lymph nodes (PLNs). DCs labelled with fluorescent cell trackers are subcutaneously (s.c.) injected in mice. After allowing the DCs to reach the PLNs, animals are intravenously injected with FLIVOTM, a permeant fluorescent reagent that selectively marks apoptotic cells. Of note, the fluorophore moiety of the latter reagent should be selected so as its emission wavelength can be distinguished under the confocal microscope from that of the fluorescent cell tracker used to label the cells. Explanted PLN are then examined under a two-photon microscope to analyse for the presence of apoptotic cells among the DCs injected. Caspases are the key proteolytic components of the demolition machinery that cleaves vital cell substrates during apoptosis1. These proteases display in their active center a cysteine residue that is required for their activity1. Active caspases can be detected in situ by Fluorochrome-Labeled Inhibitors of CAspases (FLICA)2-5, a technique based on the fact that fluorochrome-labeled inhibitors of caspases, through its conjugated reactive fluoromethyl-ketone (fmk) moiety, can form an irreversible thio-methyl ketone link with the cystein in the active center of these enzymes. Importantly, reagents based on FLICA do not bind to pro-caspases or any inactive form of these enzymes. Specifically, to detect apoptotic DCs in vivo we have used FLIVOTM, a reagent based on FLICA that is composed of a poly caspase binding inhibitor probe (Val-Ala-Asp(OMe)-fluoromethyl ketone (VAD-FMK)) which binds irreversibly to apoptotic6 caspases-1, -3, -4, -5, -6, -7, -8 and -9, conjugated to fluorescent dyes (either red SR-FLIVO or green FAM-FLIVO)7. These reagents can be intravenously injected to live mice and, since they are cell permeant, they can be used to stain apoptotic DCs in the LNs. In non-apoptotic DCs, which lack caspases, FLIVO reagents are not retained and leak out. However, in apoptotic DCs they form covalent bonds with intracellular caspases, resulting in the trapping of the (green or red) FLIVOTM fluorescent signal within these cells. Subsequently, the LNs can be explanted and examined by two photon microscopy to search for the presence of fluorescent FLIVO, indicating apoptosis, in the Dcs.Further details
The protocol was published on Protocol Exchange on 10 June 2009. To see the entire protocol, click on the source link.Advertisement
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