Flow cytometry analysis of gap junction-mediated Cel-Cell communication: Advantages and pitfalls

Paula Candida Fonseca, Oscar Kenji Nihei, Wilson Savino, David C. Spray, Luiz Anastacio Alves

Research output: Contribution to journalReview article

28 Citations (Scopus)

Abstract

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 DiIC18 (3) (10 μM) for 1h 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.

Original languageEnglish (US)
Pages (from-to)487-493
Number of pages7
JournalCytometry Part A
Volume69
Issue number6
DOIs
StatePublished - Jan 1 2006

Fingerprint

Gap Junctions
Cell Communication
Flow Cytometry
Coloring Agents
Fluorescent Dyes
Population
Immune System
Electron Microscopy
Injections

Keywords

  • Flow cytometry
  • Gap junctions
  • Intercellular communication
  • Lymphocytes

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Histology
  • Cell Biology

Cite this

Flow cytometry analysis of gap junction-mediated Cel-Cell communication : Advantages and pitfalls. / Fonseca, Paula Candida; Nihei, Oscar Kenji; Savino, Wilson; Spray, David C.; Alves, Luiz Anastacio.

In: Cytometry Part A, Vol. 69, No. 6, 01.01.2006, p. 487-493.

Research output: Contribution to journalReview article

Fonseca, Paula Candida ; Nihei, Oscar Kenji ; Savino, Wilson ; Spray, David C. ; Alves, Luiz Anastacio. / Flow cytometry analysis of gap junction-mediated Cel-Cell communication : Advantages and pitfalls. In: Cytometry Part A. 2006 ; Vol. 69, No. 6. pp. 487-493.
@article{58766e65aa2f46fe90c9f0ee98afb0a0,
title = "Flow cytometry analysis of gap junction-mediated Cel-Cell communication: Advantages and pitfalls",
abstract = "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 DiIC18 (3) (10 μM) for 1h 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.",
keywords = "Flow cytometry, Gap junctions, Intercellular communication, Lymphocytes",
author = "Fonseca, {Paula Candida} and Nihei, {Oscar Kenji} and Wilson Savino and Spray, {David C.} and Alves, {Luiz Anastacio}",
year = "2006",
month = "1",
day = "1",
doi = "10.1002/cyto.a.20255",
language = "English (US)",
volume = "69",
pages = "487--493",
journal = "Cytometry. Part A : the journal of the International Society for Analytical Cytology",
issn = "1552-4922",
publisher = "Wiley-Liss Inc.",
number = "6",

}

TY - JOUR

T1 - Flow cytometry analysis of gap junction-mediated Cel-Cell communication

T2 - Advantages and pitfalls

AU - Fonseca, Paula Candida

AU - Nihei, Oscar Kenji

AU - Savino, Wilson

AU - Spray, David C.

AU - Alves, Luiz Anastacio

PY - 2006/1/1

Y1 - 2006/1/1

N2 - 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 DiIC18 (3) (10 μM) for 1h 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.

AB - 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 DiIC18 (3) (10 μM) for 1h 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.

KW - Flow cytometry

KW - Gap junctions

KW - Intercellular communication

KW - Lymphocytes

UR - http://www.scopus.com/inward/record.url?scp=85047697917&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85047697917&partnerID=8YFLogxK

U2 - 10.1002/cyto.a.20255

DO - 10.1002/cyto.a.20255

M3 - Review article

C2 - 16646046

AN - SCOPUS:85047697917

VL - 69

SP - 487

EP - 493

JO - Cytometry. Part A : the journal of the International Society for Analytical Cytology

JF - Cytometry. Part A : the journal of the International Society for Analytical Cytology

SN - 1552-4922

IS - 6

ER -