Flowjo 10 not displaying one specific flow gat

NaГЇve T cells undergo clonal expansion and differentiation upon antigen-priming. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discriminationВ of the G 0/G 1 and G 2/M DNA peaks, while preserving membrane and intracellular staining. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G 0, in G 1 and in S-G 2/M phases of the cell cycle in the spleenВ and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.Д¬ritical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. Here, we describe a flow cytometric method for capturing a 'snapshot' of T cells in different cell cycle phases in mouse secondary lymphoid organs. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G 1 that can remain in this phase, proceed into cycling, or move into G 0. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G 0. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations.

Flowjo 10 not displaying one specific flow gat