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.
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.
Authors: Michael D Johnson; Jodi Schilz; Mirjana V Djordjevic; Jerry R Rice; Peter G Shields Journal: Cancer Epidemiol Biomarkers Prev Date: 2009-12 Impact factor: 4.254
Authors: Maria E Moreno-Fernandez; Cesar Mauricio Rueda; Paula A Velilla; Maria Teresa Rugeles; Claire A Chougnet Journal: AIDS Res Hum Retroviruses Date: 2011-11-02 Impact factor: 2.205
Authors: Martin Vaeth; Tea Gogishvili; Tobias Bopp; Matthias Klein; Friederike Berberich-Siebelt; Stefan Gattenloehner; Andris Avots; Tim Sparwasser; Nadine Grebe; Edgar Schmitt; Thomas Hünig; Edgar Serfling; Josef Bodor Journal: Proc Natl Acad Sci U S A Date: 2011-01-24 Impact factor: 11.205
Authors: Oscar K Nihei; Paula C Fonseca; Nara M Rubim; Andre G Bonavita; Jurandy S P O Lyra; Sandra Neves-dos-Santos; Antonio C Campos de Carvalho; David C Spray; Wilson Savino; Luiz A Alves Journal: BMC Cell Biol Date: 2010-01-15 Impact factor: 4.241