NK cells exist in human or animal peripheral blood, spleen, lymph nodes and bone marrow, can kill certain tumor cells, viral infection target cells, without the need for antigen or mitogen stimulation, and does not depend on antibodies or complement, in the body to kill tumors, defense against infections and play an important role in immune regulation. (Source: Basic Medical Immunology Laboratory Guide, edited by Jin Boquan and Li Enshan, 1st edition, Beijing: World Book Publishing House, 1990).
Operation method
Killing function of natural killer (NK) and lymphokine-activated killer (LAK) cells
Principle
NK cells exist in human or animal peripheral blood, spleen, lymph nodes and bone marrow, and can kill certain tumor cells, viral infection target cells without antigen or mitogen stimulation, and do not depend on antibodies or complement, and play an important role in the body's killing of tumors, defense against infections, and immune regulation.LAK is a function of NK or T cells induced by a high dose of IL-2 in vitro, which can acquire the ability to kill NK-resistant tumor cells. function, which has been widely used in the treatment of tumors by overt immunotherapy. By doping the isotope 51Cr into the target cell K562 (erythroleukemic leukemia cells) of NK or target cell LiBr (melanoma cells) of LAK, and incubating with the effector cells (NK or LAK) at a certain cell ratio for 4 hours, the killing activity of the killer cells was calculated according to the level of 51Cr released from the supernatants of the cells after the target cells were killed.
Materials and Instruments
K562 LiBr Move I. Preparation of effector cells Caveat 1. It is best to take 3-4 different effector/target cell ratios for each test, with commonly used effector/target ratios of 100:1, 50:1, 25:1, 12.5:1, and 12.5:1.The common effector/target ratios are 100:1, 50:1, 25:1 and 12.5:1. 2. pay attention to the aseptic operation in the process of inducing LAK cells. 3. avoid isotope contamination. 3. avoid isotope contamination. 4. According to the experimental needs, purified NK cells can be used as effector cells, and NK cells can be centrifuged by Percoll discontinuous density gradient.The purified NK cells can be used as effector cells according to the needs of the experiment.(1) 1 Lytic unit (LU) is 30% lysis (killing) of a certain effector cell 5×10%.3of target cells (K562) at a certain effector cell 30% lysis (killing)level.(2) If further purification of LGL is required, it can be achieved by removing goat erythroid receptor cells (T cells) with high affinity, since NKcells only have low affinity for sheep erythroid receptor (ER). This was accomplished by placing Percoll-isolated cells located in the 2nd tertiarydegree was washed and the cell concentration was adjusted to 5×106/ml, transferred to a test tube, and added 2 ml FCS and 2 ml 1 × 108The cell concentration was adjusted to 5×10 6/ml after washing.ml SRBC, centrifuged at 100 g for 5 min, incubated at 29 ℃ for 1 h, gently resuspended the cells, stacked on 3 ml Ficoll, centrifuged at 400 g for 30 min at room temperature.Cells that do not form a wreath at the 30 min interface of the center are LGL, and the purity can be >90%.(3) When using PBMC as effector cells for NK activity assay, if you want to remove the mixed erythrocytes in the PBMC suspension, you can use distilled water hypotonic method.The erythrocytes can be removed by hypotonic distilled water without using NH4Cl method, because the latter can reduce NK function.(4) In the mouse NK assay, attention must be paid to the strain and age of the mice, as there are obvious differences in NK activity between different strains of mice.(5) NK levels in mice less than 3 weeks or more than 3 months old are generally very low. For more product details, please visit Aladdin Scientific website.
3H-TdR 51Cr FCS RPMI1640 rHu IL-2
Incubation Bottles Incubation Plates CO2 Incubator Liquid Flash Meter Counter
NK-functional effector cells can be taken from resting PBMC, or PBMC induced by different cytokines.
LAK-functional effector cells usually take PBMC or tumor-infiltrating lymphocytes (TIL) or purified lymphocytes from body fluids (carcinomatous pleural and abdominal fluids) purified lymphocytes (1 × 106 /ml) from PBMC or tumor-infiltrating lymphocytes (TIL) or body fluids (cancerous pleural fluid) are usually induced by 10% FCS RPMI1640 containing a high dose of IL-2 (200-1000 μ/ml) for 2-5 days. RPMI1640 containing a high dose of IL-2 (200-1000 μ/ml) for 2-5 days.
Killing test
There are mainly 4 h 51Cr release method and 3H-TdR release method, the former requires good quality 51Cr, isotope half-life The former requires good quality 51Cr, short half-life of isotope and short operation time; the latter requires aseptic operation and longer time.
1. 51Cr 4 h release test
(1) Harvest target cells in logarithmic growth phase (K562, LiBr, etc.) After washing, adjust the cell concentration to 2×106 /ml. (2) Take 1×106 cells (0.5 ml), add Na251 CrO4 100 μci. Incubate at 37 ℃ for 2~3 h, gently shake every 15 min (3) Wash with 10% FCS RPMI1640 for 3 times, each time at 800 rpm for 5 min. (4) Resuspend the cells at a concentration of 1×105/ml. (5) Add 100 μl ( 1×104 cells) to each well of 96-well V- or U-shaped culture plate. 3 replicate wells per portion
(6) Add 100 μl of effector cells of different cell concentrations to each well, and add 100 μl of effector cells for the maximum release group. For the maximum release group, add 100 μl of 1% TritonX-100; for the natural release group, add 100 μl of complete medium. (7) Centrifuge at 200 g for 1 min. Incubate at 37 ℃ and CO2 for 4 h, and then incubate at 200 g for 1 min. 200 g centrifugation for 1 min (8) Remove 100 μl of supernatant from each well and determine the cpm value on a γ-counter.
(9) Calculation
Experimental group cpm-natural release cpm Specific killing rate (%) = ───────────── Maximum release cpm - natural release cpm
2. 3H-TdR release test
(1) Harvest target cells (K562, LiBr, etc.) in logarithmic growth phase after washing adjust the cell concentration to 2×106 /ml, add 3H-TdR 20 μci (2) Incubate at 37 ℃ for 2~3 h. (3) Wash the cells with 10% FCS RPMI1640 for 3 times, each time at 800 rpm for 5 min. Adjust the cell concentration to 1×105 /ml (4) In 96-well U-shaped or flat-bottomed culture plates, add 100 μl ( 1×104 cells) to each well. 3 replicate wells per portion (5) Add 100 μl of effector cells of different cell concentrations to each well. 100 μl of 1% TritonX-100 was added to the maximum release group, and 100 μl of complete culture medium was added to the natural release group. Natural release group was added 100 μl of complete culture medium. (6) Incubate the cells in CO2 incubator for 18~24 h. (7) Aspirate 100 μl of supernatant from each well, add trypsin (final concentration of 0.15％) and DNAzyme (final concentration of 0.0125 %) (8) Continue incubation for 30 min (9) Harvest on fiberglass paper using a cell collector (10) After drying, transfer to a liquid flash bottle, add 1 ml of scintillation solution, and β-liquid cpm was measured in the β-liquid scintillator
(11) Calculate
Experimental group cpm Specific release rate (%) = (1- ────── )×100％ Control group cpm