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Quantitation of functional T cells by limiting dilution
1University of Michigan School of Medicine, Ann Arbor, Michigan, USA.
Culturing by limiting dilution (LD) can be used to estimate the proportion of T cells in a cell preparation which can respond to an activating stimulus to produce interleukin 2 or to generate cytotoxic T lymphocyte (CTL) activity. In the first basic protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. This protocol can be modified as described in the first alternate protocol to quantitate responses to alloantigens, anti-CD3 antibody, protein antigens such as keyhole limpet hemocyanin (KLH), and viruses. The second alternate protocol provides a modification for using human responder cells. The second basic protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2.
Culturing by limiting dilution (LD) can be used to estimate the proportion of T cells in a cell preparation which can respond to an activating stimulus to produce interleukin 2 or to generate cytotoxic T lymphocyte (CTL) activity. In the first basic protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. This protocol can be modified as described in the first alternate protocol to quantitate responses to alloantigens, anti-CD3 antibody, protein antigens such as keyhole limpet hemocyanin (KLH), and viruses. The second alternate protocol provides a modification for using human responder cells. The second basic protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2.
