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 ].
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