BD Horizon RealBlue™ 705 Reagents

Normalized emission profiles of RB705, BB700 and PerCP-Cy5.5 fluorochromes. Human CD4 SK3 antibody acquired on a BD FACSDiscover^™ S8 Cell Sorter. 

Healthy PBMC were stained with a 12-color inhibitory T cell panel and acquired on the BD FACSLyric^™ Flow Cytometry System. Labels on top of each plot show the parental gate from which plots are derived; numbers in each quadrant represent the frequency of cell population; T cell subsets are identified as N, naïve; CM, central memory; EM, effector memory; EMRA, effector memory RA+; and SCM, stem cell like memory.

Peripheral blood mononuclear cells were isolated and loaded with Violet Proliferation Dye 450 before stimulation with or without staphylococcal enterotoxin B (SEB, 1 µg/mL) and CD28 (1 µg/mL) for 3 days. PMA (10 ng/mL) and ionomycin (1 µg/mL) were added to the Stimulated + Boost group 4 hours before collecting cells for staining. Cells were then stained with Fixable Viability Stain 620 and antibodies against cell surface markers prior to fixing and permeabilizing with BD Cytofix/Cytoperm^™ Fixation/Permeabilization Buffer. Fixed and permeabilized cells were then stained with intracellular antibodies. Stained cells were analyzed on a BD FACSymphony^™ A5 SE Cell Analyzer. Gating strategy for detection of T cell subsets after exclusion of doublets, dead cells and lineage-positive cells. CD3+ T cells were selected and divided into CD4+ or CD8+ cells. CD8+ T cells were further evaluated for activation stage based on their expression of CD25 and CD69. Samples were acquired on a BD FACSymphony^™ A5 SE Cell Analyzer and analyzed with FlowJo^™ Software.