Cell quest flow cytometer

Mid-log phase C. krusei cells (1×10⁶ CFU/ml), incubated with 0.8 mM CGA-N46 in 20 mM PBS (pH 6.0), were incubated for 12, 24 and 48 h at 30°C, with agitation. Subsequently, the cells were collected, washed, resuspended and fixed in 70% ice cold ethanol for 24 h. The fixed cells were then incubated with the PI solution (50 µg/ml PI, 100 µg/ml RNase (Sangon Biotech Co., Ltd.), and 0.2% Triton X-100 supplemented with PBS) for 30 min in the dark. Cell cycle distribution analysis was performed using the Cell Quest Flow Cytometer (FACScalibur; BD Biosciences), and the results were analyzed using the modFit LT™ software, version 3.1 (Verity Software House, Topsham, ME, USA).

To measure intracellular IL-17, PBMCs after culturing with PPD were stimulated with PMA (50 ng/ml) plus ionomycin (1 μg/ml) and monensin (3 μM monensin) (BD, USA) for 4 hr in 1 ml RPMI 1640 media containing 10% fetal calf serum (FCS). For surface staining, FITC anti-CD4 was added. Cells were washed with PBS containing 2% FCS and were fixed/permeabilized with %2 fixation/permeabilization buffer and stained with the anti-human IL-17PE antibody (BD, USA). After staining, cells were analyzed using CellQuest flowcytometer (BD. USA).