A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8⁺ Memory T Cells

Take non-human primate peripheral blood mononuclear cells, or PBMCs, containing CD8⁺ memory T cells against simian immunodeficiency virus or SIV.

Add a protein kinase inhibitor to prevent internalization of the T cell receptor, or TCR upon subsequent stimulation.

Introduce peptide-major histocompatibility complex class I, or pMHC-I tetramers, fluorophore-conjugated complexes comprising four MHC-I molecules bound to SIV-specific peptides. These peptides bind to TCRs on the surface of T cells.

Add a mixture containing fluorophore-conjugated antibodies binding to CD8, CD28, and CCR7 — markers delineating memory T cell subsets — and antibodies labeling non-CD8⁺ cells.

A fluorescent viability dye labels dead cells.

Fix the cells. Permeabilize them to access intracellular targets.

Add fluorophore-conjugated antibodies binding CD3, a T cell marker, and intracellular granzyme  B, an activated CD8⁺ T cell marker.

Using flow cytometry, exclude non-CD8⁺ T cells and dead cells.

Gate pMHC-I tetramer positive CD8⁺ T cells to characterize different memory CD8⁺ T cell subsets based on granzyme B, CD28, and CCR7 levels.