Briefly, CD4+ CD25− T cells (104 cells in 100 μl of medium) were seeded into a 96-well culture plate, preincubated for 60 min with nIL-2, BMS-345541, PS-1145 or vehicles, added with 20 μl of BrdU label (1 : 2000) in fresh medium, activated by the addition of MACS iBeads particles loaded with anti-CD3 plus anti-CD28 monoclonal antibodies, and maintained at 37° in a 5% CO2 humidified atmosphere for the indicated times (see results).
In controls, BrdU label was omitted. After incubation, cells were treated with fixative/denaturing solution and incubated with anti-BrdU monoclonal antibody. Unbound antibody was removed by washing and goat anti-mouse HRP-conjugate was added. Following extensive washing, fluorogenic substrate Bioactive Compound Library chemical structure was added and fluorescent product intensity
measured Ponatinib purchase at 355 nm (excitation) and 444 nm (emission) using a Fluoroskan Ascent-Thermo microplate fluorometer (Thermo Fisher Scientific, MA). Data are the ratio of the signals obtained from the labelled (BrdU) sample to those obtained from the unlabelled sample (no BrdU) after subtraction of endogenous fluorescence. For CD4 and CD25 expression analysis, cells were washed with PBS supplemented with 0·5% bovine serum albumin (BSA) (A3156; Sigma-Aldrich) and stained for 20 min at 4° with fluorescein isothiocyanate (FITC)-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-CD25 (Becton-Dickinson, Morin Hydrate NJ) and Cy-5-conjugated anti-CD3 (Caltag Laboratories, Burlingame, CA) with appropriate isotype control. Cells were washed, resuspended in PBS/BSA and analysed using an EPICS XL Beckman-Coulter, CA flow cytometer. Analysis of DNA content was carried out using propidium iodide staining. Briefly, naïve CD4+ CD25− T cells (1 × 106) were pretreated for 1 hr with DMSO, 3 μm BMS-345541 or 3 μm PS-1145 and then stimulated for 24 hr with anti-CD3 plus anti-CD28 antibodies. After treatment, cells were washed in PBS and fixed on ice with 70% volume/volume (v/v) cold ethanol to a final concentration of 65% v/v. Fixed
cells were washed in PBS, resuspended in propidium iodide (PI) solution (20 μg/ml PBS) containing DNase free RNase A (50 μg/ml PBS), incubated for 30 min at room temperature in the dark and analysed by flow cytometry.28 Cultured cells (3 × 106) were washed with PBS at 4° and extracted on ice in 50 μl of RIPA buffer [50 mm Tris-HCl, pH 7·4, 150 mm NaCl, 1% v/v Triton X-100, 0·25% weight/volume (w/v) sodium deoxycholate, 1 mm ethylenediaminetetraacetic acid (EDTA), 1 mm NaF, 1 mm Na3VO4 and 1 mm Na4P2O7] containing 1% v/v protease inhibitor cocktail. Lysate was centrifuged at 18 000 g for 5 min at 4°, and the supernatant was collected and stored at −80°. Protein concentration was determined using the DC Protein Assay kit.