Killing function is an important aspect of the body's immune function, and there are a variety of target cells with killing function in the immune system, such as natural killer cells (NK), cytotoxic T-cells (CTL), and monocyte macrophages with ADCC effects.
Operation method
Detection of natural killer (NK) cells and lymphokine-activated killing (LAK) function
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 to kill tumors, defense against infections, and immune regulation.LAK is the NK or T cells induced by a high dose of IL-2 in vitro, and obtain the function of being able to kill the 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 (erythroleukemia 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 based on the level of 51Cr released from cell supernatants after the target cells were killed.
Materials and Instruments
Cell Lines Move I. Preparation of effector cells Caveat 1. It is better to take 3~4 different effector/target cell ratios for each test, and the commonly used effector/target ratios are 100:1, 50:1, 25:1 and 12.5:1. 2. pay attention to aseptic operation during the induction of LAK cells. 3. avoid isotope contamination. 3. avoid isotope contamination.According to the experimental needs, purified NK cells can be used as effector cells, and Percoll discontinuous density gradient centrifugation is commonly used to purify NK cells (see table).1 lysis unit (Lyticunit, LU) for a certain effector cell 30% lysis (killing) 5 × 103/level of target cells (K562).For further purification of LGL, this can be done by removing sheep erythrocyte receptor cells (T cells) with high affinity, since NK cells only have low affinity for sheep erythrocyte receptor (ER). This was done by washing Percoll-isolated cells located between gradients 2 and 3 and adjusting the cell concentration to 5 × 106/ml, transferred to a test tube and added 2 ml FCS and 2 ml 1×108/ml SRBC, centrifuged at 100 g for 5 min, incubated at 29 degrees for 1 h, gently resuspended the cells, superimposed on 3 ml Ficoll, centrifuged at 400 ug for 30 min at room temperature, the cells that do not form a wreath at the interface are LGL, and the purity can be greater than 90%.When using PBMC as effector cells for NK activity assay, if you want to remove the mixed erythrocytes in the PBMC suspension, you can remove the erythrocytes by the hypotonic method of distilled water instead of the NH4Cl method because the latter method can reduce the NK function.In mouse NK experiments, attention must be paid to the strain and age of the mice; NK levels in mice within three weeks or over three months of age are generally very low. For more product details, please visit Aladdin Scientific website.
FCS RPMI 1640 51Cr
Culture flasks Culture plates CO2 incubators Centrifuges β-liquid flash γ-counter
1. NK-functional effector cells can be taken from resting PBMC, or PBMC induced by different cytokines.
2. LAK-functioning effector cells are usually formed by inducing PBMC or tumor-infiltrating lymphocytes (TIL) or lymphocytes isolated from body fluids (cancerous thymus fluid) ( 1×106/ml ) in 10% FCS RPMI 1640 containing a high dose of IL-2 (200-1000 ul/ml) for 2-5 d. The effector cells are usually induced in the same way as the lymphocytes.
II. Kill test
The main methods are the 51Cr 4 h release method, which has a short operating time and is very sensitive, and the 3H-TdR release method, which requires aseptic operation and takes a longer time.
1. 51Cr4h release test(1) Target cells (K562, LiBr, etc.) in logarithmic growth phase were harvested, washed and the cell concentration was adjusted to 2×106/ml
(2) Take 1×106 cells (0.5 ml), add Na251CrO4 100 uCi, incubate at 37 degrees for 2~3 h, and shake gently every 15 min.(3) Wash three times with 10% FCS RPMI 1640, each time at 800 rpm and 5 min.(4) Resuspension of cells at a concentration of 1 × 105/ml.(5) In 96-well v-type or U-type culture plates, add 100 ul (1×104 cells) to each well, and set up 3 duplicate wells.(6) Add 100 ul of effector cells of different cell concentrations to each well, 100 ul of 1% Triton X-100 for the maximum release group; 100 ul of complete medium for the natural release group.(7) Centrifuge at 200 g for 1 min.(8) Incubate at 37 degrees Celsius, CO2 incubator for 4 h.(9) Centrifuge at 200 g for 1 min.(10) Remove 100 ul of supernatant from each well and determine the value by β-counter.(11) Calculation: Specific kill rate (%) = (experimental cpm - naturally released cpm) / (control - naturally released cpm)
2. 3H-TdR release test
(1) Target cells (K562, LiBr, etc.) in the logarithmic growth phase were harvested, washed and the cell concentration was adjusted to 2×106/ml with 3H-TdR 20 uCi.(2) Incubate at 7 degrees for 2~3 h.(3) Wash three times with 10% FCS RPMI 1640, each time at 800 rpm and 5 min, and adjust the concentration to 1 × 105/ml.(4) In 96-well U-shaped or flat-bottomed culture plates, add 100 ul (1 × 104 cells) to each well, and set up three replicate wells.(5) Add 100 ul of effector cells of different cell concentrations to each well, 100 ul of 1% Triton X-100 for the maximal release group; 100 ul of complete medium for the natural release group.(6) CO2 incubator incubation for 18~24 h.(7) Remove 100 ul of supernatant from each well and add trypsin (final concentration 0.15%) and DNAzyme (final concentration 0.0125%)(8) Incubation was continued for 30 min.(9) Harvested on fiberglass paper using a cell collector.(10) After baking and drying, the value was determined by a beta counter.(11) Calculation: Specific kill rate (%) = (1 - experimental group cpm/control group cpm) x 100%