1. Preparation of Standard Curve
To quantitate the amount of hydrogen sulfide in a sample, A standard curve was derived from the serial dilutions of hydrogen sulfide donor, Sodium Sulfide.
 1) HSip-1 stock solution (10 mmol/l) was diluted with PBS to prepare 200 μmol/l HSip-1 working solution.
 2) Sodium Sulfide (-SulfoBiotics- Sodium Sulfide (Na₂S), 7.8 mg) were dissolved in 1 ml of de-oxygenated H₂O prepared by bubbling of nitrogen gas (100 mmol/l Na₂S solution).
 3) Na₂S solution (100 mmol/l, 20 μl) was added to 980 μl of de-oxygenated H₂O to prepare 2 mmol/l Na₂S solution.
 4) Na₂S solution (2 mmol/l,100 μl) was added to 900 μl of de-oxygenated H₂O to prepare 200 μmol/l Na₂S solution.
 5) Na₂S solution (200 μmol/l) was diluted with de-oxygenated H₂O to prepare various concentration of Na₂S solution by serial dilution (200, 100, 50, 25, 12.5, 6.3, 3.2, 0 μmol/l).
 6) HSip-1 working solution (200 μmol/l, 350 μl) was added to 300 μl of the Na₂S solutions and mixed using a vortex mixer.
 7) The solutions were incubated at room temperature for 30 minutes and 200 μl of the solution were transfered to each well (96-well plate).
 8) The fluorescence intensities were measured at 516 nm (λex=491 nm) with a microplate reader.

Fig. Fluorescence intensity changes at 516 nm with various concentrations of hydrogen sulfide.

2. Experimental Example with HeLa Cells
 1) Inoculate HeLa cells in 96-well microplate and incubate the cells in a CO₂ incubator overnight.
 2) Wash the cells with PBS buffer and remove the supernatant.
 3) Add Lysis buffer * 100 μl to a well and pipet it to lyse cells.
 4) Add HSip-1 working solution 100 μl to a well and incubate the cells at room temperature for 30 minutes.
 5) Measure the fluorescence intensity on a plate reader.( Ex: 491 nm, Em: 516 nm)
 * Lysis buffer: 6 mol/l Urea, 2% SDS, 150 mmol/l Tris-HCl (pH7.4)
The hydrogen sulfide concentration was 3 to 9 μmol/l in HeLa cells. The concentration was obtained from the standard curve, according to the fluorescence intensity of the sample.