Development of a high-throughput fluorescent no-wash sodium influx assay
Fig 7
Food dyes in the red-violet color range quench the background fluorescence of the calcium indicators fluo-4, fura-2 and fura-5F.
(A) Sample traces of the fluorescence level of SH-SY5Y cells with fluo-4 following the addition of Brilliant Black BN (1 mM), Ponceau 4R (1 mM), Allura Red (1 mM) or Amaranth (1 mM) compared to the FLIPR Calcium 4 Assay Kit obtained using the FLIPRTETRA. (B) The average relative light units corresponding to 270–300 s after addition of quenchers or FLIPR Calcium 4 Assay Kit. Statistical significance was determined using one-way ANOVA with Dunnett’s post-test, *P < 0.05 compared to wash. (C) Fluorescent response of KCl (100 mM) on SH-SY5Y cells (quantified by maximum response) in the presence of fluo-4 with washing or preincubation with FLIPR Calcium 4 Kit, Brilliant Black BN (1 mM), Ponceau 4R (1 mM), Allura Red (1 mM) or Amaranth (1 mM). Data are presented as mean ± SEM from three wells. Statistical significance was determined one-way ANOVA with Dunnett’s post-test, *P < 0.05 compared to FLIPR Calcium 4 Assay Kit. (D) Concentration-response curves for TRPV4 channel agonist GSK1016790A on MDA-MB-468 breast cancer cells obtained using FLIPR Calcium 4 Assay Kit, BD Calcium Assay Kit, or Fluo-4 with quenchers (1 mM). Fluorescent response of ATP (100 μM) on MDA-MB-468 breast cancer cells (quantified by maximum response) in the presence of (fura-5F with washing or preincubation with either (E) Ponceau 4R or (F) Brilliant Black BN. Statistical significance was determined using t-test, *P < 0.05 compared to wash. Data are presented as mean ± SEM from three wells.