Bv421 labeled streptavidin

1×10⁶ stimulated and un-stimulated PBMC per well, were treated with Brefeldin A (BD Biosciences, San Jose, CA) at 1X final concentration, incubated for 5 hours at 37°C, 5% CO₂, and assessed for TNF-alpha production via intracellular cytokine staining. PBMC were stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Life Technologies, Grand Island, NY) for 30 minutes on ice, then stained with antibodies against cell surface markers (Table 2), as described above. Cells were fixed for 10 minutes at room temperature with 1% paraformaldehyde and placed at 4°C overnight in FACS buffer. The following day, the cells were permeabilized with 1X BD FACS Perm II Buffer (BD Biosciences, San Jose, CA) at room temperature for 10 minutes, stained with biotinylated TNF-alpha (AbD Serotec, Raleigh, NC) for 20 minutes on ice. To detect the biotinylated TNF-alpha, cells were stained with BV421 labeled Streptavidin (Biolegend, San Diego, CA) for 20 minutes on ice. Acquisition and analysis was performed on a BD LSR-II using the BD DIVA software (San Jose, CA) and Flowjo Software 9.6.4 Version (TreeStar, Ashland, OR). Duplicate samples were tested.

HLA‐DRB1*01:01 restricted B. Anthracis Protective Antigen peptide 713–732 (KLPLYISNPNYKVNVYAVT) and HLA‐DRB1*04:01 restricted HIV‐1 Gag p24 peptide 164–183 (AFSPEVIPMFSALSEGATPQ) were custom synthesized to greater than 95% purity (GenScript). Peptide loaded biotinylated monomers were obtained from the National Institutes of Health Tetramer Core Facility. The peptide‐loaded monomers were subsequently cross‐linked with PE‐labeled streptavidin (Invitrogen) or BV421‐labeled streptavidin (BioLegend) for at least 4 h at 4°C to produce peptide tetramers. PBMCs (150‐250 × 10⁶) were thawed and rested at 37°C for 2 h before staining with Live/Dead Zombie viability dye. Tetramer staining was carried out for 1 h (HIV‐1) or 2 h (B. anthracis) at room temperature using 10 μg/mL of tetramer. Cell surface antibodies were added in the last 20 min of the tetramer stain. Tetramer tagged cells were magnetically labelled by adding anti‐PE beads (Miltenyi Biotec) and enriched by passing through a magnetic column (Miltenyi Biotec). Cells were acquired on a Fortessa X‐20 flow cytometer (BD Biosciences) and analyzed using FlowJo software (BD Biosciences).