Mixed lymphocyte proliferation assay

Summary

The mixed lymphocyte proliferation assay, also known as mixed lymphocyte culture (MLC) or as mixed lymphocyte response (MLR), is commonly used in tissue matching prior to organ transplantation to determine the degree of recipient and donor major histocompatibility antigen (HLA antigen) compatibility.

Principle

The basic principle of the mixed lymphocyte proliferation assay is that when two unrelated individual lymphocytes with normal function are mixed in vitro, they can stimulate each other's T-cells to proliferate due to the different HLAII antigens, which is known as two way mixed lymphocyte culture (two way MLC).

If one of the lymphocytes is treated with mitomycin C or irradiated so that the DNA in the cells loses its ability to replicate, but can still stimulate the transformation of the other lymphocytes, this is called one way MLC.

Operation method

Mixed lymphocyte proliferation assay

Materials and Instruments

Equipment: cell culture flasks, culture plates, pipettes, etc., CO2 cell culture incubator, ultra-clean table.
Reagents:
① 10% FCS-RPMI 1640 medium ; ② Cell DCs cells, reaction cells, peripheral blood mononuclear cells.
② Cells DCs cells, reaction cells, peripheral blood single nucleated cells.
③ 3 H-TdR working solution

Move

The basic procedure of the mixed lymphocyte proliferation assay can be divided into the following steps:
(I) Preparation of stimulated cells (DCs)
A. Take heparin-anticoagulated peripheral blood from healthy volunteers, and isolate single nucleated cells by lymphocyte isolation solution.
B. Suspend the cells with 10% FCS-RPMI 1640 in a culture flask and incubate them at 37℃ with 5% _CO2 for 2 hours, then gently turn the flask over to discard the supernatant, and then discard the non-adherent cells. Discard the culture supernatant, and discard the non-adherent cells to obtain the adherent monocytes
C. Add 10% FCS-RPMI 1640 and 20 ng/ml hGM-CSF, 500 IU/ml IL-4, and incubate the cells until the 7th day, then collect the suspended cells, which is the DCs
D. Stimulate the cells to lose proliferative ability by irradiating them with 25 μg/ml of mitomycin C or 2000 rad y-rays. D. Stimulated cells were treated with 25 μg/ml mitomycin C or 2000 rad y-ray irradiation so that the stimulated cells lost their proliferation ability.
(ii) Preparation of reaction cells
A Prepare monocytes from healthy human peripheral blood, wash with PBS for 3 times, wall-wrap for 2 hours at 37℃ with 5% _CO2, collect the suspended cells as reaction cells
B Resuspend the cells in RPMI 1640 culture medium and adjust the concentration of the cells to ^1x106/ml .
(iii) Mixed lymphocyte reaction
A Adjust the concentration of the stimulated cells to ^1x106, ^5x105, respectively, 2x10, 5x105, ^1x105, ^5x104 cells/ml, 10% FCS-RPMI 1640 in 96-well culture plate, set up 3 wells for each concentration, 100μL/well
B Add the concentration of reaction cells according to ^1x106 cells/ml per well into each of the above wells, 100μl/well
C Set up the stimulation cell control group (100μl of different concentration of stimulation cells + 100μl of culture medium) and reaction cell control group (100μl of different concentration of stimulation cells + 100μl of culture medium) at the same time. 100μl) and response cell control group (100μl of response cells + 100μl of culture medium)
D After incubation at 37℃ with 5% _CO2 for 5 days, the proliferation level of response cells was detected by 3 H-TdR doping rate.

Caveat

1. pay attention to aseptic operation in the experiment2. For one-way mixed lymphocyte experiments, stimulate the cells to receive a precise dose of irradiation, so that the cells temporarily survive, but lose the ability to proliferate.

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