Suitability of various membrane lipophilic probes for the detection of trogocytosis by flow cytometry.
Trogocytosis is a recently discovered phenomenon whereby lymphocytes capture fragments of the plasma membrane from antigen presenting cells (APCs). Using APCs labeled with widely used fluorescent lipophilic probes, we previously described a trogocytosis analysis protocol (TRAP) useful to understand the mechanisms and biological consequences of this process and to identify lymphocytes reacting specifically with antigen-bearing APCs. We have compared the suitability of 22 different fluorescent lipophilic probes for use in TRAP assays with cytotoxic T lymphocytes (CTL). The criteria we used were: simple and efficient incorporation in APC membranes, minimal passive diffusion among cells but efficient transfer onto T cells during trogocytosis. Sphingosin-based probes were found to incorporate inefficiently into cells. For others with unsaturated lipid chains, we found a tendency for extensive passive diffusion. In the end, about a third of the probes tested were found to be suitable in TRAP assays, which all carry either C16 or C18 saturated carbon chains, including some that can be excited with a red laser. Moreover, we found it possible to combine TRAP assays based on lipophilic probes with intracellular cytokine detection. We have identified a set of new lipophilic fluorescent probes suitable for TRAP assays in combination with intracellular staining.
Daubeuf
Sandrine
S
CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), 205 route de Narbonne, Toulouse F-31077, France.
Bordier
Christine
C
Hudrisier
Denis
D
Joly
Etienne
E
D000818:Animals; D000938:Antigen-Presenting Cells / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007371:Interferon-gamma / Q000378:metabolism; D051379:Mice; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*
D005456:Fluorescent Dyes; D007371:Interferon-gamma