A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux.
Indo-1 and high-power water-cooled lasers have been the standard for flow cytometric based Ca(2+) flux measurements. With advances in technology and the availability of low-power air-cooled lasers, there is interest in alternative protocols. Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. The reagents were examined in parallel to detect Ca(2+) flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line. Ca(2+) flux was detected with a FACSVantage SE equipped with an Omnichrome Series 74 Helium-Cadmium, or a Spectra Physics 177-G1202 Argon ion air-cooled laser. Following determination of optimal loading conditions, Ca(2+) flux was examined in response to membrane receptor stimulation or intracellular Ca(2+) mobilization. Dose dependent Ca(2+) flux to anti-CD3 and thapsigargin was detected with either Indo-1 or with fluo-3 and Fura Red. The profile of the Ca(2+) flux detected by Indo-1 or with fluo-3 and Fura Red appeared similar, with the combination of fluo-3 and Fura Red more sensitive under the particular test conditions. The results clearly demonstrated that Indo-1 could be usefully excited with a low-power air-cooled laser. The alternative use of fluo-3 and Fura Red does not require the availability of a UV capable laser and produced equivalent data.
Department of Immunology, Allergy and Arthritis, Flinders Medical Centre and Flinders University, Bedford Park, Adelaide, South Australia SA5042, Australia.
Sheree
S
Bailey
Peter J
PJ
Macardle
D000814:Aniline Compounds / Q000737:chemistry*; D001572:Benzofurans / Q000737:chemistry*; D017252:CD3 Complex / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007093:Imidazoles / Q000737:chemistry*; D007211:Indoles / Q000737:chemistry*; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry*
D000814:Aniline Compounds; D001572:Benzofurans; D017252:CD3 Complex; D005456:Fluorescent Dyes; D007093:Imidazoles; D007211:Indoles; D014966:Xanthenes; C082877:fura red; C059715:Fluo-3; D019284:Thapsigargin; C048960:indo-1; D002118:Calcium