Sulfidefluor 7-AM

Sulfidefluor 7-AM is a stable hydrogen sulphide (H₂S) fluorescent probe^[1]. Sulfidefluor 7-AM is a click chemistry reagent, it contains an Azide group and can undergo copper-catalyzed azide-alkyne cycloaddition reaction (CuAAc) with molecules containing Alkyne groups. It can also undergo strain-promoted alkyne-azide cycloaddition (SPAAC) reactions with molecules containing DBCO or BCN groups. In Vitro:Guidelines (The following is a recommended protocol. This protocol is provided as a guide only and should be modified to suit your specific needs).^[2]
1. Preparation of Dye Stock Solution
1) Weigh an appropriate amount of dried SF7-AM probe and dissolve it in anhydrous DMF to prepare a 5 mM SF7-AM stock solution.
2. Preparation of Dye Working Solution
1) Dilute a volume of the 5 mM SF7-AM stock solution to a 2.5 μM working solution in complete cell culture medium. Prepare the working solution immediately before use and mix thoroughly.
3 Specific Staining Procedure
1) Two days before the imaging experiment, passage cells into phenol red-free medium and seed onto pre-coated (if necessary) four-well Lab-Tek II glass chamber slides. Culture until the cells reach 70%-80% confluency at the time of the experiment.
2) 60 min before imaging, remove the medium from each well and add 500 μL of 2.5 μM SF7-AM working solution. Incubate the cells at 37°C, 5% CO₂ for 30 min.
3) 30 min before imaging, remove the dye-containing medium and replace with fresh medium. For the positive control wells, add an appropriate amount of Na₂S stock solution to a final concentration of 100 μM. For the negative control wells, add an equal amount of water and continue incubating at 37°C, 5% CO₂ for 30 min.
4) Place the cells on the microscope stage and image the SF7-AM using an LSM 710 laser scanning confocal microscope with a 40x objective and a 488 nm Ar laser. Quantitative analysis was performed using Zen 2010 software, using uniform upper and lower thresholds to evaluate the mean pixel intensity of each image.