Lymphocyte proliferation and differentiation is an important stage in the immune response process of the body. Therefore, the detection of lymphocyte proliferation level can be used for (1) cellular immunity research (2) clinical immune function research assay.

Operation method

Lymphocyte Proliferation Function Measurement Experiment

Principle

T cells and B cells have antigen-recognizing receptors and mitogen receptors on their surfaces, which can lead to proliferation of the corresponding lymphocyte clones under specific antigen stimulation. Phytohemagglutinin (PHA), ConA, anti-CD2, anti-CD3 McAb as polyclonal stimulants can selectively stimulate the proliferation of T cells; while anti-IgM, staphylococcal A protein-containing organisms (SAC), lipopolysaccharide (LPS, which is effective in mice) stimulate the proliferation of B cells; the Proteus Versicolor (PWM), tumor stimulant PMA have a stimulating effect on the proliferation of T and B cells. proliferation of both T and B cells. Recently, it has been found that some receptors in the VLA group of the integrin family can also activate T cells after binding with the corresponding ligands. Currently, PHA is most commonly used to stimulate PBMC, and the proliferation level of T cells is determined by morphology or tritium-labeled thymidine nucleoside (3H-TdR) incorporation rate.

Materials and Instruments

PBMC
Scintillation Solution RPMI1640 CO2 Incubator
Cell Collector β-liquid scintillator

Move

1. Aseptically isolate peripheral blood mononuclear cells (PBMC) from heparin-anticoagulated blood, wash and adjust the cell count to ^1-2x106/ml with 10% FCS RPMI1640.
2. Add to 96-well culture plate, ^1~2x105 cells/100 ul/well, set up 3 wells in each group. Add the optimal dose of PHA, 100 ul/well, and set up a negative control without PHA. If we want to observe the regulation of proliferation by cytokines (e.g. IL-2) or other stimulants (PMA), we will determine the amount to be added according to the concentration of the added factors, and the final volume of each well will be 200 ul, and then we will incubate the cells at 37 ℃, 5% CO2 for 66 h.
3. Add 0.5~1 uci 3H-TdR (50 ul) per well and continue incubation for 6~12 h.
4. collect the samples on "9999" type glass fiber filter paper with DYQ-II type multi-head cell collector.
5. Bake dry and count by β-liquid flash meter.
6. Calculation
(1) Proliferation levels are directly expressed as CPM values.

(2) It can also be expressed as a stimulation index (SI)
PHA (or experimental group) cpm-machine backgroundSI = ───────────────Negative control group cpm-machine background

Caveat

1. Pay attention to aseptic operation.

2. PHA, ConA, PWM, LPS, etc. need to find out the optimal dose or suboptimal dose before formal experiments. Different manufacturers and batch numbers of mitogens often have very different effects, such as Wellcome PHA final concentration of the optimal dose of 1-3 ug/ml, while the Guangzhou Institute of Medical Technology PHA is about 20-100 ug/ml.

3. According to the experimental needs, the optimal cell concentration, captivity time and stimulus concentration should be mapped out for lymphocytes from different sources (thymus, spleen, lymph node, tonsil, peripheral blood, etc.) of different species (human, mouse, rat, rabbit, dog, etc.).

Common Problems

Source: Basic Medical Immunology Laboratory Guide, edited by Jin Boquan Li Enshan, 1st edition, Beijing: World Book Press, 1990.

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