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...

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Bibliographic Details
Published in:Cytometry. Part A 2009-05, Vol.75A (5), p.380-389
Main Authors: Daubeuf, Sandrine, Bordier, Christine, Hudrisier, Denis, Joly, Etienne
Format: Article
Language:English
Subjects:
Animals
Antigen-Presenting Cells - metabolism
Cell Line, Tumor
Cell Membrane - metabolism
Flow Cytometry - methods
Fluorescent Dyes - chemistry
fluorescent lipophilic probes
immune response
immunomonitoring
Interferon-gamma - metabolism
lymphocyte
membrane probes
Mice
T-Lymphocytes, Cytotoxic - metabolism
TRAP assay
trogocytosis
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Summary: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. © 2008 International Society for Advancement of Cytometry
ISSN:1552-4922
1552-4930
DOI:10.1002/cyto.a.20679