Measurement of T Cell Activation After 16‐hr In Vitro Stimulation with Concanavalin A

A flow cytometry assay that can be used to directly determine the proportion of activated T lymphocytes in human whole blood samples after stimulation with concanavalin A is presented here. Human whole blood is incubated with fluorescently labeled antibodies (against CD3, CD4, CD8, and CD69), erythrocytes are then lysed, and the samples are analyzed using a flow cytometer. The assay presented is able to differentiate between CD4+ and CD8+ T lymphocytes. Thus, it is possible to quantify both lymphocyte populations in parallel, as well as the respective proportions of activated T lymphocytes, all from one sample. An additional advantage of this assay is that it was developed to assay whole blood, thereby reducing the likelihood of introducing pre‐analytic error and facilitating the calculation of quantitative ratios. Thus, the method can be used for both in vitro and ex vivo experiments. Curr. Protoc. Cytom. 51:6.28.1‐6.28.10. © 2010 by John Wiley & Sons, Inc.