Design of a multicolor panel to assess intracellular and surface molecules by flow cytometry

Introduction: Flow cytometry allows simultaneous detection of surface and intracellular molecules on each cell. Objective: To describe a method for building up a harmonic multicolor panel with 11 flow cytometry parameters for phenotypic and functional analysis on CD8(+) T lymphocytes. Materials and methods: For the multicolor panel construction, we selected the molecules and titred conjugated antibodies with fluorochromes for CD3, CD8, CCR7, CD28, CD27, CD45RA, CD95 and CD127 determination in peripheral blood mononuclear cells (PBMC). To evaluate the panel, the conjugated antibodies were gradually added one by one and fluorescence minus one (FMO) test was performed. This method was applied to assess ex vivo subpopulations of T cells and the production of intracellular IFN gamma, IL-2 and TNF alpha using polyclonal stimulation with enterotoxin B from Staphylococcus aureus (SEB) and antigen-specific cells with crude Trypanosoma cruzi antigen. Finally, the ex vivo CD8(+) T lymphocyte subpopulations frequencies were analyzed in healthy individuals. Results: The evaluation of the selected molecules and conjugates did not show interference in the fluorescence signals and detection. The frequencies of CD8(+) T cells evaluated were similar to the values reported in other studies. Additionally, we observed that the frequency of CD8(+) T lymphocytes producing IFN gamma, IL- 2 and TNF alpha was higher 6 hours after culture with SEB and crude T. cruzi lysate. Conclusions: The method used for the construction of a multicolor panel allows obtaining frequencies of CD8(+) T lymphocyte subpopulations corresponding to those reported in the literature.