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Blastogenesis: ³H-Thymidine Incorporation

What this assay tells you

The first response of B and T lymphocytes to antigen signals is proliferation into a clone of specific cells. ³H-Thymidine is a precursor of DNA and is incorporated into new DNA in proliferating cells.

What you need to do the assay

• Antigen-stimulated lymphocytes (remember T cells must be stimulated with specific peptide on self MHC-bearing APC or target cells).
• Tissue culture supplies.
• ³H-Thymidine (thymidine in which H has been replaced by radioactive ³H (tritium).

How the assay is done

• Stimulate the lymphocytes with specific antigen under conditions where they should be activated to divide. Maintain in culture for 2-4 days until many cells are proliferating.
• Add ³H-Thymidine to the culture medium for 4-6 hours.
• Centrifuge the cells and count the amount of ³H-Thymidine using a beta spectrometer to detect the radioactivity. DNA may be isolated from the cells to ensure that the ³H-Thymidine is in the DNA.
• Negative controls eliminate the antigen stimulation. Positive controls use mitogens: chemicals that stimulate lymphocytes to divide. Common mitogens are LPS for B cells and plant-derived Concanavalin A (Con A) or Phytohemagglutinin (PHA) for T cells.

How to interpret the results

The higher the amount of ³H-Thymidine incorporated into the DNA, the more proliferation is occurring.

Additional comments

Mixed lymphocyte reactions (MLR) are used to measure T cell responses to foreign MHC antigens for transplant tissue typing. Stimulator APC from the donor, treated so they can't divide, are mixed with responder T cells from the recipient. Several days later, the culture is pulsed with ³H -Thymidine and incorporation by the responder cells is measured. The higher the radioactivity incorporated into the T cells, the stronger is the response to the foreign cells and the more likely rejection will occur.

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