A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca2+ flux

Indo-1 and high-power water-cooled lasers have been the standard for flow cytometric based Ca2+ 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 Ca2+ flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line.Ca2+ 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, Ca2+ flux was examined in response to membrane receptor stimulation or intracellular Ca2+ mobilization.Dose dependent Ca2+ flux to anti-CD3 and thapsigargin was detected with either Indo-1 or with fluo-3 and Fura Red. The profile of the Ca2+ 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.