Self-supplied consumables:
Microplate reader or visible spectrophotometer (can measure the absorbance at 520 nm)
thermostatic box, ice machine, low temperature centrifuge
96-well plate or microglass cuvette, adjustable pipette gun and tip
deionized water
Homogenizer (if it is a tissue sample)
Reagent preparation
Ferrous Salt
ready-use type; Before use, equilibrate to room temperature; Store away from light at 4 ° C.
H2O2: ready-to-use type; Before use, balance to room temperature; Store away from light at 4 ° C.
Salicylic Acid
ready-to-use; Before use, balance to room temperature; Store away from light at 4 ° C.
Sample preparation
- Animal tissues: About 0.1g of sample was weighed, 1 ml of deionized water was added, homogenized in an ice bath, centrifuged at 10,000 g for 10 min at 4 ° C, and the supernatant was taken to be measured.
- Plant tissue: weigh about 0.1g sample, add 1mL deionized water to mashed, ultrasonic crushing in an ice bath for 5 min (power 20% or 200W, ultrasonic 3 s, interval 7 s, repeated 30 times), centrifuged at 10,000 g at 4℃ for 10 min, and take the supernatant to be tested.
3, cells or bacteria: Collect 5 million cells or bacteria into a centrifuge tube, wash the cells or bacteria with cold PPBS, discard the supernatant after centrifugation, add 1mL deionized water, break the cells or bacteria in an ice bath for 5 min (20% power or 200W, ultrasonic 3 s, interval 7 s, repeated 30 times), and then centrifuge at 10,000 g at 4 ° C for 10 min. The supernatant was taken for testing.
4, serum (plasma) and other liquids with high protein content or turbidity: Take 0.1mL sample, add 1mL deionized water to mix, centrifuge at 10,000 g for 10 min at 4 ° C, and take the supernatant to be tested.
5, honey and urine and other low protein content and clear liquid: direct determination.
6, extracts or drugs: can be prepared into a certain concentration, such as 0.5 mg/mL for determination.
Experimental steps:
1, microplate reader or visible spectrophotometer preheat for more than 30 min, adjust the wavelength to 520 nm, visible spectrophotometer with deionized water to zero.
- Sample determination (successively add the following reagents to 96-well plate or microglass cuvette) :
| Reagent | Blank tube（μL） | Standard tube（μL） | Detector tube（μL） | Control tube（μL） |
|---|
| Ferrous Salt | 40 | 40 | 40 | 40 |
| H2O2 | 0 | 40 | 40 | 0 |
| Deionized water | 120 | 80 | 40 | 80 |
| Salicylic Acid | 40 | 40 | 40 | 40 |
| Sample | 0 | 0 | 40 | 40 |
| The mixture was mixed and the reaction was carried out at 37 ° C for 20 min, and the absorbance at 520 nm was measured. ΔA test =A test -A control, ΔA standard =A standard -A blank (blank and standard Wells only need to be measured once) | The mixture was mixed and the reaction was carried out at 37 ° C for 20 min, and the absorbance at 520 nm was measured. ΔA test =A test -A control, ΔA standard =A standard -A blank (blank and standard Wells only need to be measured once) | The mixture was mixed and the reaction was carried out at 37 ° C for 20 min, and the absorbance at 520 nm was measured. ΔA test =A test -A control, ΔA standard =A standard -A blank (blank and standard Wells only need to be measured once) | The mixture was mixed and the reaction was carried out at 37 ° C for 20 min, and the absorbance at 520 nm was measured. ΔA test =A test -A control, ΔA standard =A standard -A blank (blank and standard Wells only need to be measured once) | The mixture was mixed and the reaction was carried out at 37 ° C for 20 min, and the absorbance at 520 nm was measured. ΔA test =A test -A control, ΔA standard =A standard -A blank (blank and standard Wells only need to be measured once) |
Note
In order to compare the hydroxyl radical scavenging ability of different samples, the same batch of samples must be diluted in the same way, and the extract or drug must be prepared in the same concentration.
The results were calculated as follows:
The calculation formula was
hydroxyl radical clearance D%=(ΔA standard -ΔA measured)÷ΔA standard ×100%
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