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.
Keywords: activated T cells; lymphocytes; lymphocyte proliferation assay; activation marker; CD4 T helper cells; CD8 cytotoxic T cells; CD69 activation marker; T lymphocytes; CD69; CD4 T cells
Table of Contents
- Strategic Planning
- Basic Protocol 1: Immunophenotypic Analysis of the Relative Distribution of Activated and Non‐Activated CD4 and CD8 T Lymphocyte Subsets in Unwashed Erythrocyte‐Lysed Whole Peripheral Blood Samples
- Literature Cited
Basic Protocol 1: Immunophenotypic Analysis of the Relative Distribution of Activated and Non‐Activated CD4 and CD8 T Lymphocyte Subsets in Unwashed Erythrocyte‐Lysed Whole Peripheral Blood Samples
Figure 6.28.2 Antibody staining of an unstimulated sample. This sample shows CD4‐positive and CD8‐positive populations, but very few CD69‐positive T cells.
Figure 6.28.3 Isotype control staining of an unstimulated whole blood sample. CD4‐ and CD8‐positive populations are absent. This experiment demonstrates the lack of nonspecific binding.
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