Inhibition And Production Of Superoxide Anionic Colorimetric Assay Kit (WST-1 Method)

Detection principle Superoxide anion free radicals are produced through the reaction system of xanthine and xanthine oxidase. WST-1 (a water-soluble tetrazolium salt) can react with the generated superoxide anion to produce water-soluble formazan. When the tested sample contains the superoxide anion free radical inhibitor, it can inhibit the formation of formazan. When the tested sample contains the substance that produces superoxide anion free radical, it can promote the formation of formazan dye. By colorimetric analysis of WST-1 products, the units of activity of inhibition or production of superoxide anion radical in samples can be calculated. Performance characteristics Sample type Serum,plasma,urine,cells,cell culture supernatant,leucocyte Detection method Colorimetric method Assay type Activity Assay time 35 min Precision Average inter-assay CV: 5.8%Average intra-assay CV: 2% Other instruments required Micropipettor, Multi-channel pipettor, Incubator, Vortex mixer, Centrifuge Other reagents required Normal saline (0.9% NaCl), PBS (0.01 M, pH 7.4) Storage Reagent 3: -20 °C, others: 2-8 °C Valid period 6 months Dilution of sample Before the formal experiment, it needs to choose 2-3 samples for diluting a series of diluent and determine the dilution factor when the SOD inhibition ratio is 30%~65% (the optimal inhibition ratio is the range of 40%~60%). Inhibition ratio=[(A1-A2)-(A5-A6)]×100%/(A1-A2) Adjust sampling volume: If inhibition ratio > 65%, need to dilute the sample or decrease the sampling volume than take the test. If inhibition ratio < 30%, need to increase the sampling volume. The recommended dilution factor for different samples is as follows (for reference only): Sample type Dilution factor Human serum 4-7 Mouse serum 15-25 Rat serum 25-35 Human saliva 1 HepG2 culture supernatant 1 10% Rat brain tissue homogenate 150-200 10% Rat liver tissue homogenate 500-600 10% Mouse liver tissue homogenate 500-600 10% Mouse heart tissue homogenate 150-200 10% Epipremnum aureum tissue homogenate 20-30 Note: The diluent is normal saline (0.9% NaCl) or PBS (0.01 M, pH 7.4).