-SulfoBiotics- HSip-1 DASB22 Product Manual

It has been recognized that hydrogen sulfide (H₂S) has an important role as a physiological active substance for vasodilation, cytoprotection, and modulation of insulin secretion. H₂S is considered as a gaseous molecule such as nitric oxide and carbon monoxide. However, around 80% of the total sulfide exists as hydrogen sulfide anion (HS-) under physiological condition, since the pKa is about 7. In addition, HS- easily converts to various biochemical molecules such as persulfides and polysulfides, which react with sulfhydryl moieties in a living body. -SulfoBiotics- HSip-1 DA is cell membrane permeable and it enables fluorescent imaging of intracellular H₂S.

-SulfoBiotics- HSip-1 DA

50 μg x 1

 

Store at -20oC.

• Dimethyl sulfoxide (DMSO)
• Serum-free medium
• HBSS
• Micropipettes

Preparation of 1 mmol/l HSip-1 DA stock solution

Add 62 μl of DMSO to a tube containing 50 μg of HSip-1 DA and dissolve it by pipetting.

• Store at -20 °C. The reconstituted solution is stable at -20oC for 1 month.

Fluorescence imaging of hydrogen sulfide with HSip-1 DA

1. HeLa cells were seeded on μ-slide 8 well (Ibidi) and cultured at 37oC overnight in a 5% CO₂ incubator.
2. The culture medium was discarded and the cells were washed with a serum-free medium (MEM) twice.
3. HSip-1 DA stock solution (1 mmol/l) was diluted with a serum-free medium (MEM) to prepare 5 μmol/l HSip-1 DA working solution.
   • Please optimize the final concentration of HSip-1 DA depeneding on the cell lines.
4. HSip-1 DA working solution (5 μmol/l, 200 μl) was added to the cells, and the cells were cultured at 37oC for 30 minutes in a 5% CO₂ incubator.
5. The supernatant was discarded, and the cells were washed with HBSS twice.
6. Na2S solution (200 μmol/l, 200 μl) was added to the each well, and the cells were cultured at 37oC for 30 minutes in a 5% CO₂ incubator.
7. The supernatant was discarded and the cells were washed with HBSS twice.
8. HBSS (200 μl) were added, and the cells were observed by confocal fluorescence microscopy.

Fig.3 Detection of hydrogen sulfide using HSip-1 DA in HeLa cells treated with Na₂S.
(A: Control, B: 200 μmol/l Na₂S treated)

These products were commercialized under the advisory of Dr. Tetsuo Nagano and Dr. Kenjiro Hanaoka (The University of Tokyo).

1. K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami, Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura, and T. Nagano, “Development of a Highly Selective Fluorescence Probe for Hydrogen Sulfide”, J. Am. Chem. Soc., 2011, 133, 18003.