Updated: September 25, 2019

For research use only

Table 1. Product information
Catalog no.

Product nameAmountA402-1SSip-1 DA

60 nmol × 3

Storage upon receipt

Stability≤−20°C, keep desiccated1 year (when unopenedand protected from light.

and stored as described.)

1.
Intracellularly abundant sulfane sulfur species including persulfide (R-S-SH), polysulfide (R-S-Sn-S-R) andpolysulfide (H2Sn) play important roles to maintain intracellular reducing environments. SSip-1 DA is a FRETbased fluorescence probe to specifically detect the intracellular sulfane sulfur species. It shows only week
fluorescence without sulfane sulfur, and reversibly fluoresces in response to micromolar concentrations ofsulfane sulfur. Its reaction is highly specific and has minimal to no reactivity with H2S, cysteine residues, andsulfur oxides. SSip-1 DA, a cell permeable diacetylated (DA) form of SSip-1 is hydrolyzed by intracellularesterases to generate SSip-1 which retains within the cells. Thus it is suitable to monitor intracellularconcentration of sulfane sulfur via live-cell imaging.

Figure 1. Absorbance (left) and fluorescence (right) of SSip-1 in 0.1 M phosphatebuffer (pH=7.4) in the absence and presence of Na2S4.
■ Storage
Upon receipt, store the product desiccated and protected from light at ≤ −20°C. Storing as a solution is notrecommended.

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■ Materials required but not provided
・Dimethyl sulfoxide (DMSO)
・A protein solution such as bovine serum albumin (BSA)・Observation buffer (Hank’s balanced salt solution (HBSS), etc.). It should be a solution without phenol red.
■ Preparation of reagent
SSip-1 DA is a violet solid. Before opening the cap, warm the vial to the room temperature. Then, use a microcentrifuge to spin down the solid that might adhere on the cap. Add 60 μL of DMSO to one vial to prepare 1mM solution. Finally, dissolve the solid entirely by pipetting for more than five times. SSip-1 DA solution will
become red-violet.
※SSip-1 DA may aggregates in an aqueous solution. We recommend to dilute SSip-1 DA-DMSO
solution with aqueous buffer solution containing proteins such as 1 mg/ml BSA.

■ Detection of intracellular sulfane sulfur in A549 cells
1.

Prepare a cell-staining solution by diluting 1 mM SSip-1 DA solution to 10 µM with observation buffer
containing 10 mg/mL BSA.
※ Fluorescence of SSip-1 DA is diminished by reduced glutathione (GSH). Intracellular GSH
concentration is varied among different kinds of cells. Therefore, we recommend optimizing dye
concentrations and incubation time in your conditions. In GORYO Chemical, Inc., incubating A549
cells with 1 µM dye solution at 37°C for 60 minutes gave good results.
※ We recommend paying attention to intracellular nutrition because the intracellular GSH concentration
depends on the nutritional conditions.

2.

Remove culture media from the dish and rinse twice with the observation buffer.

3.

Add the cell-staining solution to the dish and incubate at 37°C for 60 minutes.

4.

After staining, wash twice with the observation buffer.

5.

Observe with a fluorescence microscope. You may induce the production of sulfane sulfur by adding
Na2S4 to the final concentration of 5 µM.

■ Fluorescence observation
Use 488- or 495-nm laser for the laser microscopy. Maximum emission is detected at 525 nm. For fluorescencemicroscopy, blue-excitation filter sets for GFP or for FITC are appropriate.
■ References
See also the publications below, for other usages and protocols.
D. Ezeriņa, Y. Takano, K. Hanaoka, Y. Urano, T. P. Dick (2018) Cell Chem. Biol. 25: 1–13
R. Miyamoto, S. Koike, Y. Takano, N. Shibuya, Y. Kimura, K. Hanaoka, Y. Urano, Y. Ogasawara, H. Kimura (2017) Sci.
Rep., 7: 45995
Y. Takano, K. Hanaoka, K. Shimamoto, R. Miyamoto, T. Komatsu, T. Ueno, T. Terai, H. Kimura, T. Nagano, Y. Urano(2017) Chem. Commun., 53: 1064-1067

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EAREE Bldg. 5F, Kita 8 Nishi 18-35-100, Chuo-ku, Sapporo 060-0008, JAPANTEL: +81-11-624-5860
https://goryochemical.com/

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