ABSTRACT
Immunocompetent cells require continued proliferation and differentiation for self-renewal and protection of the host against pathogens. One way to evaluate the proliferation of leukocytes is the lymphoproliferative response of B and T cells to mitogens. A mitogen is a compound that can induce mitosis in leukocytes. This procedure describes the mitogen-induced lymphoproliferative response in various vertebrate species. Phytohemagglutinin (PHA) and concanavalin A (Con A) are mitogens known to activate the proliferation of T cells, lipopolysaccharides (LPS) to induce blastogenesis in B cells, and pokeweed mitogen (PWM) to activate DNA synthesis in both T and B cells. During mitosis induced by a mitogen, the cells will incorporate the radioactive base 3H-methyl thymidine. The radioactivity is measured with a β counter and the results are expressed in counts per minute (CPM) or disintegrations per minute (DPM) to give a measure of proliferation, since DPM and CPM values are proportional to the level of 3H-methyl thymidine incorporated in cells (see Figure 10.1). Principle of the mitogenic assay. Lymphocytes are incubated, with or without mitogens (e.g., Con A) and with or without xenobiotics (for toxicological studies), in a microplate. Following an appropriate incubation period, tritiated thymidine is added to the wells and the cells are incubated again to allow the incorporation of the radioactive thymidine into the DNA of dividing cells. The DNA is then captured onto glass fiber papers and the radioactivity is measured with a β scintillation counter. https://s3-euw1-ap-pe-df-pch-content-public-p.s3.eu-west-1.amazonaws.com/9780429156977/936bbeac-659b-4503-8a0d-08a54ad61dfe/content/fig10_1.tif"/>
