An Assay for Cell Cycle Analysis of Antigen-Specific CD8+ T Cells
Trascrizione
Take secondary lymphoid organ cells from an antigen-immunized mouse, containing antigen-specific CD8+ T cells in different cell cycle stages.
G1, S, G2, and M are stages of actively dividing cells, while cells in the G0 stage are quiescent.
Add a fluorescent viability dye to label dead cells, and fluorophore-tagged antibodies binding to T cell surface markers CD3 and CD8.
Introduce fluorophore-conjugated antigenic peptide-MHC multimers to bind T cell receptors, labeling the antigen-specific T cells.
Using a buffer, fix and permeabilize the cells.
Introduce fluorophore-tagged antibodies to bind a cell proliferation-associated nuclear protein — staining the actively dividing cells.
Add a fluorescent stain to label DNA.
Using flow cytometry, select the living cells and identify the antigen-specific CD8+ T cells via the surface-bound labels.
The DNA stain intensity discriminates G0 and G1 cells with a lower DNA amount from the S, G2, and M cells with a higher DNA amount. The absence of the nuclear protein-specific antibody separates the G0 cells.
