Fig 1.
Schematic diagram illustrating the principle of a fluorescent wash (left) and no-wash (right) ion influx assay.
Fig 2.
Absorbance spectra of food dyes in the red-violet color range.
(A) Schematic diagram of a color wheel illustrating the emission wavelengths (inner circle) and absorption wavelengths (outer circle) of visible light (in nanometers; nm). The inner circle represents the emission color of the indicator dye and the outer circle represents the color of the quencher required to absorb the emission. For example, ANG-2 has a peak emission wavelength of 540 nm (green), therefore it can be expected that red-violet colored dyes will quench the emission (B) Absorbance spectra of Brilliant Black BN, Carmine, Ponceau 4R, Allura Red and Amaranth, Calcium 4 Assay and BD Quencher. Grey shaded area represents the FLIPRTETRA detection range (FDR). (C) International Numbering System for Food Additives (INS) E number, common name(s), color and peak absorption (λ max) of food dyes tested.
Fig 3.
Food dyes in the red-violet color range quench the background fluorescence of the sodium indicator ANG-2.
(A) Sample traces of the fluorescence levels of NaV1.7-HEK cells with ANG-2 and PSS buffer addition, with and without washing, obtained using the FLIPRTETRA. (B) Sample traces of the fluorescence level of NaV1.7-HEK cells with ANG-2 following the addition of Brilliant Black BN (1 mM), Carmine (1 mM), Ponceau 4R (1 mM), Allura Red (1 mM) or Amaranth (1 mM) obtained using the FLIPRTETRA. (C) The average relative light units corresponding to 60–100 s after addition of quenchers or buffer. Reduction in background fluorescence by Brilliant Black BN (1 mM), Carmine (1 mM), Ponceau 4R (1 mM), Allura Red (1mM) and Amaranth (1 mM) was not significantly different to washing. Statistical significance was determined using one-way ANOVA with Dunnett’s post-test, *P < 0.05 compared to wash. (D-H) Concentration-response curves for the reduction in background fluorescence of NaV1.7-HEK cells with ANG-2 by quenchers Brilliant Black BN, Carmine, Ponceau 4R, Allura Red and Amaranth. Data are presented as mean ± SEM from three wells.
Fig 4.
Ponceau 4R quenches background fluorescence of the sodium dye indicator ANG-2 without affecting NaV channel function.
(A) Fluorescent response of PSS buffer or veratridine (60 μM) at NaV1.7 (quantified by AUC) in the presence of ANG-2 with washing or preincubation with Brilliant Black BN (1 mM), Carmine (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 using t-test, *P < 0.05 compared to buffer control. (B) Sample traces of the fluorescence response obtained using ANG-2 with Ponceau 4R (1 mM) at NaV1.7 stimulated with veratridine (60 μM) from three wells. (C) Sample traces of the fluorescence response obtained using ANG-2 with Ponceau 4R (1 mM) at NaV1.8 stimulated with deltamethrin (150 μM) from three wells. (D) Representative NaV1.7 current trace before (dashed) and after (solid) addition of Ponceau 4R (1 mM) obtained from whole-cell patch clamping. (E) I-V curve of NaV1.7 with Ponceau 4R (1 mM; white squares) compared to time-matched buffer control (black squares) (F) G-V curve of NaV1.7 with Ponceau 4R (1 mM; white squares) compared to time-matched buffer control (black squares). Voltage-dependence of steady-state fast inactivation at NaV1.7 with Ponceau 4R (1 mM; white circles) compared to time-matched buffer control (black circles). Data are presented as mean ± SEM, n = 4–7 cells.
Fig 5.
Concentration-response curves for veratridine at hNaV1.1–1.7 and deltamethrin at hNaV1.8 obtained using a no-wash sodium influx assay using ANG-2 and the quencher Ponceau 4R.
Addition of the non-selective NaV channel activator veratridine concentration-dependently increased fluorescence with the following EC50’s: NaV1.1 21 μM, NaV1.2 16 μM, NaV1.3 12 μM, NaV1.4 16 μM, NaV1.5 23 μM, NaV1.6 10 μM, NaV1.7 29 μM. Addition of the NaV channel activator deltamethrin concentration-dependently increased fluorescence at NaV1.8 with an EC50 of 169 μM. Data are presented as mean ± SEM from three wells.
Fig 6.
Concentration-response curves for the NaV channel inhibitor tetracaine at hNaV1.1–1.8 obtained using a no-wash sodium influx assay ANG-2 and the quencher Ponceau 4R.
Preincubation of tetracaine concentration-dependently reduced the fluorescence response caused by addition of veratridine (60 μM; NaV1.1–1.7) or deltamethrin (150 μM; NaV1.8) with the following IC50’s: NaV1.1 52 μM, NaV1.2 44 μM, NaV1.3 36 μM, NaV1.4 13 μM, NaV1.5 14 μM, NaV1.6 66 μM, NaV1.7 21 μM, NaV1.8 6 μM. Data are presented as mean ± SEM from three wells.
Table 1.
Potency of veratridine, deltamethrin and tetracaine on NaV1.1-NaV1.8 determined using the 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.