Flow cytometry analysis of gap junction-mediated cell-cell communication: advantages and pitfalls.
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 microM) for 30 min at 37 degrees C, and the other population was incubated with the lipophilic dye DiIC(18) (3) (10 microM) for 1 h at 37 degrees 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.
Fonseca
Paula Candida
PC
Laboratório de Comunicação Celular, Departamento de Imunologia, Instituto Oswaldo Cruz, Fundação, Rio de Janeiro, Brasil.
Nihei
Oscar Kenji
OK
Savino
Wilson
W
Spray
David C
DC
Alves
Luiz Anastacio
LA
D000818:Animals; D002232:Carbocyanines / Q000378:metabolism / Q000494:pharmacology; D002450:Cell Communication / Q000502:physiology; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism*; D006801:Humans; D007365:Intercellular Junctions / Q000378:metabolism*; D008954:Models, Biological
D002232:Carbocyanines; D005452:Fluoresceins; D005456:Fluorescent Dyes; C024286:3,3'-dioctadecylindocarbocyanine; C007740:fluorexon