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).

1. 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:

3, blending, immediately read at 450 nm absorbance value of A0, room temperature (25 ℃) avoid light incubation 60 min, again read at 450 nm absorbance value A60, as computing delta A = A60 - A0, respectively as for delta A empty, ? A v, ? A measurement, Calculate ??A pair =?A pair -?A empty, ??A test =?A test -?A empty.

The results were calculated as follows:

The ultra oxygen anion clearance calculation formula: D = % as as (delta delta as as A v - delta delta A test) as as present delta delta A x 100%

Superoxide anion (O2 -) is a transient free radical created by adding electrons to oxygen. It is formed by environmental factors such as ultraviolet light, cigarette smoke, environmental pollutants and gamma radiation, or it is derived from oxidases such as xanthine oxidase or NADPH oxidase. O2 - Once formed, it attacks cellular components and damages lipids, proteins and DNA. This triggers a variety of diseases, including cancer, atherosclerosis, rheumatoid arthritis, diabetes, liver damage, and central nervous system disease. More and more attention has been paid to scavenging superoxide anion free radicals. The superoxide anion scavenging capacity detection kit (micromethod) can detect the superoxide anion scavenging capacity of animal and plant tissues, cells, blood, drugs and other samples. The principle is that xanthine oxidase (XO) catalyzes the reaction to provide superoxide anion (O2 -). O2 - reacts with the tetrazolium salt WST-8 dye to form a water-soluble colored methylene dye product, which can be readily quantified. The sample is cleared of O2 -, so less O2 - is available for the chromogenic reaction. This scavenging ability of the sample was measured by colorimetry at OD450 nm.