Mixed lymphocyte culture (MLC) or mixed lymphocyte reaction (MLR) was previously used for tissue matching prior to organ transplantation to determine the degree of recipient and donor major histocompatibility antigen (HLA antigen) compatibility. Because lymphocytes in MLC undergo activation and proliferation when they are stimulated by isotype antigens, and produce a large variety of cytokines that promote the differentiation of killer cells such as NK, LAK, and CTL, MLC is also a commonly used experimental model in immunomodulation studies. (Source: Laboratory Guidelines for Basic Medical Immunology, edited by Jin Boquan and Li Enshan, 1st edition, Beijing: World Book Publishing House, 1990).

Operation method

Mixed lymphocyte culture

Principle

When two unrelated individual lymphocytes with normal function are mixed in vitro, they can stimulate each other's T cells to proliferate due to different HLA class II antigens, which is called two way MLC; if one of the lymphocytes is treated with mitase C or irradiated so that the DNA in the cells loses its ability to replicate, it can still stimulate the other lymphocyte to transform, which is called one way MLC. If one of the lymphocytes is treated with mitogen C or irradiated so that the DNA in the cells loses its ability to replicate, but still stimulates the other lymphocyte to transform, this is called one way MLC. The greater the difference in HLA antigens between the two individuals, the stronger the response, and the proliferation level of the responding cells can be detected by cell number, morphology, or 3H-TdR incorporation rate.EBV-transformed B lymphocytes express a high level of HLA class II antigens, and are often used as the stimulating cells in one-way MLC.

Materials and Instruments

Stimulated Cell N23 Cell Line
FCS RPMI 1640 medium
Cell Culture Flasks Culture Plates Drip Tubes Suction Tubes CO2 Incubator Ultra Clean Table

Move

1. Preparation of stimulating cells

Commonly used stimulating cells are EBV-transformed B lymphocytes (e.g., N23 cells) or PBMC.

(1) N23 cells in logarithmic growth phase are taken, centrifuged and resuspended in fresh complete medium.

(2) Adjust the cell number to ^{1×106 /ml~2×106 /ml} , and transfer to a plastic culture bottle or 50 ml centrifuge tube.

(3) Irradiate the cells at 60 ℃ for 3000 rad.
2. Preparation of reaction cells

Separate and purify the PBMC of the individual to be examined.
3. LC

(1) Resuspend 4 ml of 10% FCSRPMI1640 in the ratio of ^2×106 PBMC to ^1×105 N23 cells irradiated by 3000 rad.

(2) Place in small culture flasks for MLC, keep the flasks upright and do not shake for 4 d of incubation.

(3) Add 1 ml of fresh medium on the 5th day.

(4) If you want to measure the incorporation rate of ^3H-TdR, you can usually do it on the 5th day of MLC.

5. To detect NK, LAK or specific killer T-lymphocytes (CTL) in MLC, harvest effector cells around 7~10 d.

Caveat

1. Pay attention to aseptic operation.

2. Stimulating cells should receive the irradiation dose accurately, so that the cells temporarily survive but lose the ability to proliferate.

3. RAJI or Daudi cells can be used as stimulating cells, or a mixture of different cells can be used as stimulating cells.

4. The appropriate ratio of different stimulating cells to responding cells varies, and pre-experiments should be done to select the best experimental conditions.

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