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