Rat anti mouse igg1 v450

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 ].

To determine the location of GCR expression in CD28+ and CD28null T and NKT-like cells differential staining of whole blood following stimulation (as described above) using reagents to sequentially permeabilise the cytoplasm and nucleus as previously described [17 ]. Briefly, following stimulation, 350 μL aliquots of stimulated whole blood were treated with FACSLyse as described above and following centrifugation cell cytoplasmic membrane was permeabilised with 0.1% saponin for 10 mins. Following centrifugation, cells were resuspended in 100 μL 0.1% saponin then labeled with anti-GCR as described above. Following washing in 0.1% saponin, cells were stained with rat anti-mouse IgG1 V450 (BD) for 10 min. After washing in 0.1% saponin, the cells were permeabilised with 500 μL 0.1% Triton in PBS for 10 min. Cells were then incubated with anti-GCR as described above, followed by rat anti-mouse IgG1 PE for 10 min. After washing in 2 mls 0.5% BSA in FACSFlow, cells were stained with 5 μL of appropriately diluted CD3 perCP.Cy5.5 (BD), CD28 PECY7 (BD), CD56 APC (Beckman Coulter), CD8 APCH7 (BD) and CD45 V500 (BD) for 10 min. After washing, data was acquired as described above.