An Assay for Cell Cycle Analysis of Antigen-Specific CD8⁺ T Cells

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.

Prepare fresh 1X permeabilization wash buffer by diluting 10X permeabilization buffer with distilled water, and filter it through a 0.45-micr