Antigen-specific T cell clone preparation assay

Summary

The antigen-specific T cell clone preparation assay can be used to: prepare antigen-specific T cell clones.

Operation method

cycle stimulation

Principle

T-lymphocytes are derived from pluripotent stem cells in the bone marrow (and from the yolk sac and liver in the embryonic stage). During the embryonic and neonatal stages of the human body, a portion of the pluripotent stem cells or pre-T cells from the bone marrow migrate to the thymus gland, where they are induced by thymic hormones to differentiate and mature into immunologically active T cells.

Materials and Instruments

MNC Antigen
RPMI 1640 culture medium
24-well culture plate 37℃ constant temperature box 96-well culture plate

Move

I. Materials and reagents
1. Antigen-sensitized donor single nucleated cells (MNCs) and 3,300 rad irradiated own MNCs
2. Antigens
3. Complete RPMI 1640 culture medium with 10% human AB serum
4. Other reagents and equipment required for cell isolation and T-cell culture and proliferation
II. Operational steps
1. Antigen stimulation: Add ^5×106/2 ml/well of mononuclear cells and the optimal concentration of antigen to a 24-well culture plate, and incubate at 37℃ for 7 d in a 5% _CO2 incubator.
2. Intermittent cyclic stimulation:
(1) Resuspend antigen-stimulated cells and subject to density gradient centrifugation, collect cells in the interfacial layer and wash them twice with serum-free RPMI-1640;
(2) Adjust the cell concentration to ^1×106 /ml and add it to the wells of 24-well culture plate, meanwhile, add irradiated self MNC ^4×106 to each well, and use unirradiated self cells as feeder cells and antigen-presenting cells, and incubate for 7d;
(3) Collect and wash the cells as in step (1);
(4) The cell concentration was adjusted to 1 × ¹⁰⁶ /ml and co-cultured with 2 to 3 × ¹⁰⁶ freshly irradiated self MNCs and antigens for 7d;
(5) Repeat steps (2) to (4) for a total of 3 cycles;
(6) Cells are collected and antigen specificity is determined by a proliferation assay.
3. T-cell cloning: The cells cultured by intermittent cycling stimulation were diluted to a limited extent, and cell suspensions of different concentrations were added sequentially to the 96-well plate, while ¹⁰⁴ irradiated own MNC, IL-2 30 U and appropriate concentrations of antigens were added to the wells of the cloning plate, and cultured for 7-10 d under the same conditions as described above, and the growth of the cells was observed under the microscope. The well-grown antigen-specific cells were transferred to the wells of 24-well plates, and self-feeder cells and antigen were added, and the culture was continued for 7 d. The cells were passaged and cultured by intermittent stimulation.

Caveat

1. T cell clones prepared by the above method do not distinguish between CD4+ and CD8+ T cells, both of which may be cloned.

2. The amount of IL-2 should not be too high before limited dilution, otherwise non-specific proliferation of T cells may occur.

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