Superoxide Anion Scavenging Capacity Test Kit (Micromethod)

Self-supplied consumables:
Microplate reader or visible spectrophotometer (measuring absorbance at 450 nm)
96-well plate or microglass cuvette, pipetting gun and tip
Ice machine, low temperature centrifuge
Deionized water
Homogenate (if it is a tissue sample)
Reagent preparation:
Assay Buffer: ready-to-use; Before use, balance to room temperature; The samples were stored at 4 ° C.
The Sample Solution: ready-to-use; Before use, balance to room temperature; The samples were stored at 4 ° C.
WST - 8: ready-to-use; Avoid light place experiments, the ice; Partial shipments - 20 ℃ avoid light preservation.
Enhancer: ready-to-use; Avoid light place experiments, the ice; Aliquots were stored at -20 ° C in the dark.
Working Xanthine Oxidase: shake well before dilution; In clean centrifuge tube by Sample Diluent as positive control and Sample requirements for 20 times dilution. Avoid light place experiments, the ice; Aliquots were stored at -20 ° C.
Xanthine: ready-to-use; The experimental process, placed on the ice; Aliquots were stored at -20 ° C. Xanthine may appear cloudy. Before moving fluid tube vortex.
Working Reagent: in 96 - well plates, each hole to 85 (including L Working Reagent, 74 mu L Assay Buffer, 5 mu L Xanthine, 5 WST - 8 and 1 mu mu L L Enhancer, is now active.

Sample preparation:
1. Animal tissues: After washing the tissues with PBS precooled on ice, all erythrocytes were removed, and the tissues were homogenized with 1 mL/0.1 g precooled lysate (50 mM potassium phosphate, 0.1 mM EDTA, 0.5% Triton X-100) and centrifuged at 12,000 g for 5 min at 4 ° C. The supernatant was removed and placed on ice until measured.
2. Plant tissue: About 0.1 g of the sample was weighed, 1 mL of precooled lysate (50 mM potassium phosphate, 0.1 mM EDTA, 0.5% Triton X-100) was added and mashed, and the cells were disrupted by sonication in an ice bath for 5 min (20% power or 200 W, 3 s, 7 s interval, repeated 30 times). After centrifugation at 12,000 g for 5 min at 4 ° C, the supernatant was removed and placed on ice until measured.
3, Cell samples: 5×10 6 cells were collected, cells were washed with precooled PBS, centrifuged and the supernatant was discarded. Cells were resuspended in 1 mL of ice-cold lysis buffer (50 mM potassium phosphate, 0.1 mM EDTA, 0.5% Triton X-100) and incubated on ice for 10 min, followed by centrifugation at 12,000 g for 5 min at 4 ° C. The supernatant was removed and placed on ice until measured.
4, a blood sample: use the standard procedures to collect serum or plasma (heparin, citric acid salt or EDTA). Erythrocyte precipitates could be dissolved in 5 times the volume of ice-cold deionized water. 12000 g centrifugal 5 min to precipitate the red cell membrane. Before the ultra oxygen anion clearance test, using the Sample Diluent dilution serum/plasma (5), red blood cell cracking liquid (1:100).
5, drug: diluted to a certain concentration. Such as 1 mg/mL.
Note: The following substances should be avoided in sample preparation: ascorbic acid, sodium azide, > 0.2% SDS, > 1% NP-40 and > 1% Tween-20. If not analyzed immediately, samples can be stored at − 80 ° C for up to one month. All samples are available at pH 7.4 50 mM dilute potassium phosphate.

Experimental procedures:
1. The microplate reader or visible spectrophotometer was preheated for more than 30 min, and the wavelength was adjusted to 450 nm. The visible spectrophotometer was zeroed with deionized water.
2. Add the following reagents to a 96-well plate or microglass cuvette: