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Flow cytometry analysis of gap junction‐mediated cell–cell communication: Advantages and pitfalls
Author(s) -
Fonseca Paula Candida,
Nihei Oscar Kenji,
Savino Wilson,
Spray David C.,
Alves Luiz Anastacio
Publication year - 2006
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20255
Subject(s) - flow cytometry , gap junction , population , fluorescence , cytometry , biophysics , chemistry , materials science , nanotechnology , biomedical engineering , biology , microbiology and biotechnology , intracellular , physics , medicine , optics , environmental health
Background: Since the first morphological description of the gap junctions use electron microscopy, a considerable number of techniques has been introduced to evaluate gap junction channel functionality, many of which use dye transfer techniques, such as dye injection and fluorescent dye transfer, analyzed by flow cytometry. Methods: To analyze dye transfer, generally one population of cells is incubated with calcein‐AM (0.5 μM) for 30 min at 37°C, and the other population was incubated with the lipophilic dye DiIC 18 (3) (10 μM) for 1 h at 37°C; after incubation, these cells were washed five times with PBS and cocultured for different times, and then the dye transfer was analyzed by flow cytometry. Results: In this short overview, we focus on some advantages and disadvantages of flow cytometry as a technique to investigate gap junction‐mediated intercellular communication (GJIC). In addition, we point out some technical pitfalls that we have encountered when applying this technique to study gap junctions in immune system cells. Conclusions: Analysis of fluorescent dye transfer by flow cytometry is a useful tool to investigate GJIC. However, some points must be taken into consideration before using this methodology, which are discussed herein. © 2006 International Society for Analytical Cytology