The source of this experiment is the official website of the Fourth Military Medical University

Operation method

NK cell activity by lactate dehydrogenase (LDH) release assay

Principle

Lactate dehydrogenase (LDH) is one of the enzymes contained within the cytoplasm of living cells. Under normal conditions, it cannot pass through the cell membrane. When the target cell is damaged by the attack of effector cells, the permeability of the cell membrane is changed, and LDH can be released into the medium. The released LDH catalyzes the generation of pyruvate from lactic acid by turning oxidized coenzyme I (NAD+) into reduced coenzyme I (NADH2), and the latter then passes through the transmitter of hydrogen-phenoxazinedimethyl ester sulfate (PMS) reducing iodine nitro-nitrozolium chloride blue (INT) or nitro Tetrazolium Blue Chloride (NBT) to form colored methylzine compounds, which have a high absorption peak at 490 nm or 570 nm, and NK cell activity can be calculated by using the OD value of the readings.

Move

1. Target cell preparation

Take the target cells cultured for 24-48h, wash them 3 times, and finally adjust the cell concentration to 1×105/ml with complete RPMI-1640 culture medium, and set aside.2. Preparation of effector cells

PBMC or mouse splenocytes were isolated by conventional methods, washed 3 times, and finally the cell concentration was adjusted to 1×l07/ml with complete RPMI-1640 culture solution.3. Efficacy-target cell action

Add 0.1ml of effector cells and target cells (E/T=100:1) into the wells of 40-well cell culture plates, set up 3 replicate wells for each specimen, and set up a target cell natural release control group and a maximum release control group (0.1 ml of target cells + 0.1 ml of 1% NP-40 solution), centrifuge at a low speed of 1000r/min for 2min, and then place in the incubator at 37℃, 5% CO2 for incubation. 2h.4. Enzymatic reactions

Remove the culture, aspirate 0.1ml of supernatant from each well and add it to another well of the culture plate, preheat at 37℃ for 10min, add 0.1ml of freshly prepared LDH substrate solution to each well, and react for 10~15min at room temperature and protected from light, and then add 30μl of 1 mol/L citric acid termination solution to terminate the enzymatic reaction in each well.5. Calculation of results

The OD value of each well was read at 570 nm with an enzyme-linked detector and NK cell activity was calculated.NK cell activity (%) = ■ × 100%

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