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.
Arneth
Borros M
BM
Institute of Clinical Chemistry and Laboratory Medicine, Johannes Gutenberg University Mainz, Mainz, Germany.
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D002457:Cell Extracts; D003208:Concanavalin A / Q000276:immunology* / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D008213:Lymphocyte Activation / Q000187:drug effects; D016131:Lymphocyte Subsets / Q000187:drug effects; D013997:Time Factors
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D003208:Concanavalin A