Free H2S was measured in cells using the SF7-AM fluorescent probe31 (link) (Sigma-Aldrich). The probe was dissolved in anhydrous DMF at 5 mM and used at 5 μM in serum-free RPMI medium with or without VSMCs. Free polysulfide was measured in cells using the SSP4 fluorescent probe. The probe was dissolved in DMF at 10 mM and diluted at 10 μM in serum-free RPMI medium with or without VSMCs. Fluorescence intensity (λex = 495 nm; λem = 520 nm) was measured continuously in a Synergy Mx fluorescent plate reader (BioTek Instruments AG, Switzerland) at 37 °C before and after addition of various donors, as indicated.
Plasma polysulfides were measured using the SSP4 fluorescent probe. Plasma samples were diluted 3 times and incubated for 10 min at 37 °C in presence of 10 μM SSP4. Plasma polysulfides were calculated using a Na2S3 standard curve. Liver polysulfides were measured using the SSP4 fluorescent probe. Pulverized frozen liver was resuspended in PBS-0.5% triton X-100, sonicated and adjusted to 0.5 mg/ml protein concentration. Lysates were incubated for 30 min at 37 °C in presence of 10 μM SSP4 and fluorescence intensity (λex = 495 nm; λem = 520 nm) was measured in a Synergy Mx fluorescent plate reader (BioTek Instruments AG, Switzerland)
Plasma polysulfides were measured using the SSP4 fluorescent probe. Plasma samples were diluted 3 times and incubated for 10 min at 37 °C in presence of 10 μM SSP4. Plasma polysulfides were calculated using a Na2S3 standard curve. Liver polysulfides were measured using the SSP4 fluorescent probe. Pulverized frozen liver was resuspended in PBS-0.5% triton X-100, sonicated and adjusted to 0.5 mg/ml protein concentration. Lysates were incubated for 30 min at 37 °C in presence of 10 μM SSP4 and fluorescence intensity (λex = 495 nm; λem = 520 nm) was measured in a Synergy Mx fluorescent plate reader (BioTek Instruments AG, Switzerland)