NK cell activity assay: LDH method
NK cell activity assay: LDH method
This experiment was mainly used to determine NK cell activity.
Operation method
NK cell activity assay: LDH method
Principle
The cytoplasm of living cells contains LDH.Under normal conditions, LDH cannot pass through the cell membrane, and when the cells are killed by NK cells, LDH is released outside the cell.LDH can dehydrogenate lithium lactate, which in turn reduces NAD to NADH, which is then reduced to iodine nitro tetrazolium chloride (INT) via the hydrogen delivery body phenazine dimethyl sulfate (PMS), which accepts H+ to be reduced to a purplish red color. Methyl-Luna-zan analogs. Determined colorimetrically on an enzyme marker at 490 nm.
Materials and Instruments
YAC-1 cells Move 1. Preparation of LDH substrate solution Lithium lactate 5×10-2mol/L Nitrotetrazolium chloride (INT) 6.6×10-4mol/L Phenazine dimethyl sulfate (PMS) 2.8×10-4mol/L Oxidized coenzyme I (NAD) 1.3×10-3mol/L The above reagents were dissolved in 0.2 mol/L Tris-HCl buffer (pH 8.2) 2、Target cell passaging (YAC-1 cells) The target cells were passaged and cultured 24h before the experiment. The cells were washed 3 times with Hank's solution before application, and the cell concentration was adjusted to 4×105 cells/mL with RPMI1640 complete culture solution. 3、Preparation of spleen cell suspension (effector cells) Spleen was taken aseptically and placed in a small flat dish containing appropriate amount of sterile Hank's solution, and the spleen was gently ground with tweezers to make single cell suspension. The spleen was filtered through a 200-mesh sieve, or ground up with 4 layers of gauze, or washed twice with Hank's solution, and centrifuged for 10 min (1000 r/min) each time. The supernatant was discarded to eject the cytoplasm, and 0.5 mL of sterilized water was added for 20 s. After lysing the erythrocytes, 0.5 mL of 2x Hank's solution and 8 mL of Hank's solution were added, centrifuged at 1000 rpm for 10 min, and the cells were resuspended with 1 mL of RPMI 1640 complete culture medium containing 10% calf serum and diluted with 1% glacial acetic acid diluted and counted (the number of live cells should be above 95%), the number of live cells was counted by staining with Formosan (should be above 95%), and finally the cell concentration was adjusted to 2×107 cells/mL with RPMI1640 complete culture solution. 4、NK cell activity detection Take target cells and effector cells 100 μL each (effect-target ratio 50:1), and add them into U-type 96-well culture plate; add target cells and culture medium 100 μL each to the target cell natural release well, and target cells and 1% NP40 or 2.5% Triton 100 μL each to the target cell maximal release well; set up three re-wells for each of the above items, and cultivate them for 4h in 37℃ and 5% CO2 incubator, then incubate the 96-well culture plate at 1500 r/l at 1500 r/l. Then, centrifuge the 96-well culture plate at 1500r/min for 5min, and put 100μL of supernatant into the flat-bottomed 96-well culture plate, meanwhile, add 100μL of LDH substrate solution, and then react for 3min, and then add 30μL of 1 mol/L HCl into each well, and then measure the optical density value (OD) at 490nm in the enzyme marker. The NK cell activity was calculated according to the following formula, the NK cell activity of the tested sample group was significantly higher than that of the control group, and the result of the experiment could be judged as positive. OD of reaction wells - OD of natural release wells NK cell activity (%) = ────────────────── × 100% Maximum release hole OD - natural release hole OD Data processing and result determination NK cell activity needs to be converted to data, X = Sin-1 , where P is NK cell activity, expressed in decimals, and then analyzed by ANOVA, in the analysis of variance, it is necessary to perform ANOVA procedures in accordance with the ANOVA chi-square test first, variance chi-square, calculate the F-value, F-value < F0.05, the conclusion: the difference between the means of each group is not significant; F-value ≥ F0.05, P ≤ 0.05 with two-by-two comparison method of means between multiple experimental groups and one control group for statistics; appropriate variable transformation for data that were not normal or variance-aligned, and after meeting the requirements of normal or variance-aligned, the transformed data were used for statistics; if the variable transformation still did not meet the purpose of normal or variance-aligned, the rank-sum test was used for statistics instead. Caveat 1. target cells and effector cells must be fresh, and the cell viability should be more than 95%. 2. The ambient temperature should be kept constant during colorimetry. 3. LDH matrix solution should be prepared before use. 4. Within a certain range, the NK cell activity is proportional to the effector-target ratio. Generally, the target ratio should not exceed 100. For more product details, please visit Aladdin Scientific website.
Hank's Liquid RPMI1640 Complete Culture Solution Lithium Lactate Sodium Lactate Nitrotetrazolium Chloride Phenazine Dimethyl Sulfate Tris-HCl Buffer NP-40 Triton NAD Kit
Enzyme marker Tweezers Sieves Gauze Centrifuge Culture plates