Tetramer Staining of Cryopreserved NHP PBMCs

Cryopreserved PBMCs from BCG-vaccinated NHP were thawed in RPMI 50% FCS and plated at a concentration of 3×10⁶ cells/well in a 24 well plate for 2 to 4 hours for monocyte plastic adhesion. Non-adherent cells were collected and stained with 1:800 dilution of viability dye (Thermofisher) for 10 minutes at 4°C. Cells were washed once with FACS buffer (PBS 0.1% BSA) and divided to be stained with 1:50 dilution of different tetramers for 15 minutes at 37°C. HLA-E tetramers were produced and peptide loading was confirmed by mass spectrometry as described previously (Ruibal et al. submitted) (18 (link)). Antibodies for CD3 (clone SP34–2, BD Biosciences) and CD8 (clone RPA-T8, BD Biosciences) staining were added and incubated for 30 minutes at 4°C. Cells were washed once as before, fixed with 1% paraformaldehyde and immediately acquired on a FACSLyric (BD Biosciences). All data were analyzed using FlowJo software v10.7.1 (BD Biosciences).