Reagents and equipment:
1. Peroxide storage solution: 30mmol / L hydrogen peroxide stored in T-bovine serum albumin (Tris-bovine serum albumin solution);
2. Sample buffer: 2 volumes of T-bovine serum albumin solution + 1 volume of 2% (volume fraction) Triton X-100;
3. Titanium oxysulfide (2.25g / L) is dissolved in 1mol / L sulfuric acid;
4. T-bovine serum albumin: 20 mmol/L Tris-HCl (pH 7.0) containing 1 g/L bovine serum albumin;
5. Microcentrifuge with 2.0ml microcentrifuge tube;
6. Spectrophotometer;
experimental method:
After primary cell culture, all operations were carried out in 2 ml microcentrifuge tubes on ice and all solutions were maintained in an ice bath environment.
1. Freshly prepare the mixture to be tested, and dilute 8.5 ml of the peroxide storage solution to 100 ml with T-bovine serum albumin solution;
2. Add 1.0 ml of titanium oxysulfide to 0.5 ml of the assay mixture and measure the absorbance at a wavelength of 405 nm, which should be about 1.5. If not, adjust the concentration of hydrogen peroxide accordingly;
3. Mix 10 μl of sample with 30 μl of sample buffer, and use 40 μl of sample buffer for reaction control;
4. Add 0.5ml of the mixture to be tested to all tubes, and only 6 tubes per batch;
5. After accurately counting for 1 min, add 1.0 ml of titanium oxysulfide compound and centrifuge at 13500 g for 2 min with a microcentrifuge;
6. All tubes were measured for absorbance at 405 nm after equilibration at room temperature. The blank contained 0.54 ml T-bovine serum albumin and 1 ml titanium oxysulfide compound;
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