Dojindo,硫生物-HSip-1/1/SB21

Fig. 2 Excitation and emission spectra of HSip-1 reacted with H₂S(Em: 491nm/Ex: 516nm)

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.

Quantification of Hydrogen Sulfide in HeLa cells:1. Preparation of Standard CurveTo 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) 用 PBS 稀释 HSip-1 储备溶液 (10 mmol/l) 以制备 200 μmol/l HSip-1 工作溶液。2) 将硫化钠 (-SulfoBiotics- Sodium Sulfide (Na2S), 7.8 mg) 溶解在 1 ml通过氮气鼓泡制备的脱氧 H2O（100 mmol/l Na2S 溶液）。3) 将 Na2S 溶液（100 mmol/l，20 μl）加入 980 μl 脱氧 H2O 中，制备 2 mmol/l Na2S 溶液.4) 将 Na2S 溶液 (2 mmol/l,100 μl) 添加到 900 μl 脱氧 H2O 中，制成 200 μmol/l Na2S 溶液。5) 用脱氧 H2O 稀释 Na2S 溶液 (200 μmol/l)通过连续稀释（200、100、50、25、12.5、6.3、3.2、0 μmol/l）制备各种浓度的 Na2S 溶液。6）将 HSip-1 工作溶液（200 μmol/l，350 μl）加入到300 μl Na2S 溶液并使用涡旋混合器混合。7) 将溶液在室温下孵育 30 分钟，然后将 200 μl 溶液转移到每个孔（96 孔板）中。8) 测量荧光强度在 516 nm (λex=49 1 nm）与酶标仪。

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

2. Experimental Example with HeLa Cells1) 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.