A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all which possess some limitations. Bromodeoxyuridine (BrdU) marks that are or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter after division. However, these dyes do not allow identification the phase at time analysis. An alternative approach is to exploit Ki67, marker highly expressed phases except quiescent G0. Unfortunately, Ki67 does further differentiation as it separate S-phase committed mitosis from those G1 can remain this phase, proceed into cycling, move Here, we describe flow cytometric method for capturing "snapshot" different mouse secondary lymphoid organs. combines DNA staining with major histocompatibility complex (MHC)-peptide-multimer an innovative gating strategy, allowing us successfully differentiate between CD8 G0, S-G2/M spleen draining lymph nodes mice vaccination viral vectors carrying model antigen gag human immunodeficiency virus (HIV)-1. Critical steps were choice dye strategy increase assay sensitivity include activated/proliferating would have missed current criteria dye, Hoechst 33342, enabled obtain high-quality discrimination G0/G1 G2/M peaks, while preserving membrane intracellular staining. great potential knowledge about response improve immuno-monitoring