Isoflow

Analyses were performed on three Cytomics FC500 flow cytometers (Beckman-Coulter, Villepinte, France). To limit background noise from dust and crystals, all three instruments were operated using 0.22-μm filtered sheath fluid (Isoflow™; Beckman-Coulter). CXP ACQUISITION and CXP ANALYSIS software packages (Beckman-Coulter) were used for data acquisition and analysis, respectively. Arabidopsis protoplasts of leave 5 were immersed in 5 μM FDA (Sigma; in MES buffer, pH 6.1) for 20 min at room temperature in the dark, and then washed three times with MES buffer (pH 6.1). Cells were stained with Annexin V using the Annexin V-FITC fluorescence detection kit (BD Biosciences, San Jose, CA, USA), in accordance with the manufacturer's instructions. Briefly, cells cultured on cover slips, and then washed twice with PBS. The slides were examined and photographed with a Nikon Eclipse TE 2000 U motorized inverted microscope (Nikon Corp., Tokyo, Japan). The apoptotic index was calculated as the percentage of cells stained positive for Annexin V. A total of 100 cells were counted in each experimental group in three independent experiments and results arethe mean proportion of apoptotic cells in sixscanning electron micrographs.

Production of IFNγ and TNFα by T, NKT-like and NK cells was determined on blood and BAL samples from all subjects as previously reported [11 (link)–13 (link)].
Briefly, one mL blood diluted 1:2 in RPMI and prepared BAL cells were stimulated with phorbol myristate (25 ng/mL) (Sigma, Sydney, Australia) and ionomycin (1 μg/mL) (Sigma). Brefeldin A (10 μg/mL) was added as a “Golgi block” (Sigma) and the tubes re-incubated in a humidified 5% CO₂/95% air atmosphere at 37°C for 16 h. Aliquots of blood and BAL were added to FACS tubes (BD) and treated with FACSLyse and FACSPerm as above and five μL of appropriately diluted anti- IFNγ FITC (BD), CD3 perCP.CY5.5 (BD), CD56 APC (Beckman Coulter, Sydney, Australia), CD8 APC.CY7 (BD), TNFα V450 (BD) and CD45 V500 (BD) monoclonal antibodies were added for 15 min in the dark at room temperature. Two mL of 0.5% bovine serum albumin (Sigma) / Isoflow (Beckman Coulter) was then added and the tubes centrifuged at 300 ×g for 5 min. After decanting, cells were analyzed as above.

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 ].