Anti cd19 pacific blue

PBMC were prepared as described above, re-suspended in R10 medium at 10⁶ cells per ml and distributed into 5 ml FACs tubes (Invitrogen, UK) at 10⁶ cells per tube; one tube per stimulation condition. Stimuli were added to each tube as appropriate (R10 medium as a negative control; PPD at 20 μg/ml; SEB at 5 μg/ml; FEC peptides at 25 μg/ml) and samples were incubated at 37°C for 2 h. After this time, 3 μl of brefeldin A (BFA, Sigma, UK; stock concentration 1 mg/ml) was added to all tubes to give a final concentration of 3 μg/ml, and tubes were incubated for a further 18 h (overnight) at 37°C. Following stimulation, PBMC were washed in FACS buffer (PBS with 0.1% bovine serum albumin (Sigma) and 0.01% sodium azide (Sigma)) and stained with VIVID live/dead reagent (Molecular Probes) as well as with a surface stain cocktail of antibodies (anti-CD4-APC-Cy7 (Biolegend); anti-CD14-Pacific Blue (Invitrogen); anti-CD19-Pacific Blue (eBiosciences)). After further washing, PBMC were permeabilised with Cytofix/Cytoperm reagent (BD Biosciences) and stained with an intracellular antibody cocktail (anti-CD3-PerCP (Biolegend); anti-CD8-FITC (Biolegend); anti-IFNγ-PE (Caltag)) prior to a final wash and re-suspension in 1% paraformaldehyde. Cells were acquired within 24 h of staining.