Flow Cytometry: This is a common method used to assess the expression of activation markers on the surface of T cells, such as CD69, CD25, and HLA-DR. By tagging these markers with fluorescent antibodies, researchers can measure their levels using a flow cytometer, which analyzes each cell’s characteristics.

Cytokine Production Assays: Activated T cells release cytokines like IFN-γ, IL-2, and TNF-α. Enzyme-linked immunosorbent assays (ELISA), intracellular cytokine staining, or multiplex bead assays can measure these cytokines in cell culture supernatants or within cells.

Phospho-protein Analysis T-cell activation often leads to proliferation. This can be measured using methods like carboxyfluorescein diacetate succinimidyl ester (CFSE) staining, where the dye is diluted as cells divide. Proliferation can also be measured by [³H]-thymidine incorporation or BrdU (bromodeoxyuridine) uptake assays.

Calcium Flux Assay: T-cell receptor (TCR) engagement leads to an increase in intracellular calcium, which can be measured using calcium-sensitive fluorescent dyes like Fura-2. An increase in fluorescence indicates T-cell activation.

Gene Expression Analysis: Activated T cells change their gene expression profiles. Quantitative PCR (qPCR) or RNA sequencing can be used to measure the upregulation of activation-related genes, such as IL2, IFNG, or CD25.