Facs perm

Following stimulation as described above, 350 μL aliquots of cells were treated with 2 mL FACSLyse (BD Bioscience, Sydney, Australia) for 10 min. Cells were centrifuged, supernatant discarded and 500 mL FACSPerm (BD) added for 10 min. Two mL 0 · 5% bovine serum albumin (BSA) (Sigma) in IsoFlow (Beckman Coulter, Sydney, Australia) was then added and the tubes centrifuged at 300 g for 5 min. After decanting supernatant, Fc receptors were blocked with 10 mL human immunoglobulin (Intragam, CSL, Melbourne, Australia) for 10 min at room temperature. Five μL of mouse anti-human GCR (clone 5E4, Serotec, Sydney, Australia; raised against a conserved sequence of the regulatory part of the receptor- amino acids 150–176) as previously reported [16 (link)] was added to cells for 15 min, and following washing (as above), 5 μL rat anti-mouse IgG1 V450 (BD) was added for 15 min. Following washing, 5 μL of appropriately diluted CD3 perCP.Cy5.5 (BD), Pgp1 PE (BD), CD28 PECY7 (BD), CD56 APC (Beckman Coulter), CD8 APCH7 (BD) and CD45 V500 (BD) were added for 15 min in the dark at room temperature. Cells were washed and events acquired and analyzed as previously reported [11 (link),13 ].

Frozen PBMC were thawed and seeded in 96 well plates at a density of 0.5–1 x 10⁶/well. Cells were activated with 10 μg/ml Mtb H37Rv lysate (BEI Resources, Manassas, VA, USA) and 5 μg/ml PHA (Remel, Thermo Fisher Scientific, Lenexa, KS, USA) for 16 hours. Along with stimulants, 1X monensin (BD Biosciences, USA) and 1X Brefeldin (BD Biosciences, USA) solution and 1 μg/ml BD Fast Immune CD28/49d (BD Biosciences, USA) were also added. Unstimulated cells were used as controls. At the end of 16 hrs, cells were first stained with an antibody cocktail comprising Avid, anti-CD45RA APC-H7, anti-CD127 PerCP Cy5.5, anti-HLA-DR FITC and anti-CD25 BV421 at 4°C for 10 minutes. Cells were then fixed with 1X FACS Lyse (BD Biosciences, USA), permeabilised with 1X FACS Perm (BD Biosciences, USA) and stained with an antibody cocktail of anti-CD3 BV570, anti-CD4 BUV395, anti-IFNγ Alexa Fluor 700 (clone B27, BD PHArmingen, BD Biosciences, USA), anti-IL-2 PE (clone MQ117H12, BD Biosciences, USA), anti-IL17A BV605 (clone BL168, Biolegend, San Diego, CA, USA), anti-IL-22 PECy7 (clone 22URT1, eBiosciences, San Diego, CA, USA) and anti-IL-10 BV786 (clone JES3-9D7, BD Biosciences, USA) at room temperature for 30 minutes. Cells were washed, fixed with 1% paraformaldehyde and acquired on BD FACS Aria Fusion using appropriate compensation controls. Data was analysed using FlowJo.