Cd19 brilliant violet 510

Experimentally infected monocytes were coincubated with CD8⁺ T cells from the same, seropositive donor overnight in the presence of CD107a Alexa Fluor 647, 5 μg/ml brefeldin A, and 2 μM monensin (all from BioLegend) at 37°C. CD8⁺ T cells were harvested and washed and then stained with a combination of surface antibodies (CD3 brilliant violet 650, CD14 brilliant violet 510, and CD19 brilliant violet 510; BioLegend) and LIVE/DEAD Fixable Aqua Dead cell stain (Invitrogen) at 4°C. Cells were fixed and permeabilized using FIX & PERM (ADG, Kaumberg, Austria) and stained intracellularly with antibodies (CD69 Pacific Blue, 4-1BB phycoerythrin [PE]-Cy5, CD8 brilliant violet 570, and granzyme A FITC [BioLegend]; granzyme B FITC [Miltenyi Biotec, Inc.]; granzyme K FITC [Santa Cruz Biotechnology, TX, USA]; TNF-α brilliant ultraviolet 395 and IFN-γ brilliant violet 786). Responding CD8⁺ T cell populations were identified by the expression of CD69 and 4-1BB at levels above the background, and expression levels of CD107a, TNF-α, and IFN-γ were then measured. In all cases, cell doublets, monocytes, B cells, and dead cells were eliminated from the analyzed populations.
To analyze differentiation markers, monocytes were labeled with anti-CD14 and anti-CD83 antibodies (BioLegend) (both allophycocyanin [APC] conjugated).

Thawed PBMC were rested for 12 hours prior to stimulation of 1.5 million cells each with Brefeldin A (Sigma Aldrich) and either gag peptides (1 ug/peptide/mL; NIH AIDS reagent program); Staphylococcal enterotoxin B (SEB; 1 ng/mL) or Dimethyl sufoxide (DMSO) for 6 hours. Cells were then surface stained with CD3 AlexaFluor700, CD8 Pacific Blue, CCR7 PE-CF594, PD-1 PE-Cy7 (all BD Biosciences), CD4 Qdot 605 (Invitrogen), CD45RA Brilliant Violet 650, CD19 Brilliant Violet 510 (Biolegend), CD27 APCe780, and aqua fluorescent reactive dye (Invitrogen), permeabilised with Saponin and stained intracellularly with IL-2 PerCP-Cy5.5, IFNγ APC and TNFα Alexa Fluor 488 (all BD Biosciences) prior to fixation. Cells were acquired within 24 hrs using a BD LSR-II and analysed using FlowJo version 9 and 10.