Cyan adp flow cytometry

The HHUA and AN3CA cells were treated with 0.3 µM MLN 4924 or DMSO for 48 h,
harvested and fixed in 70% ethanol at –20 °C overnight, and stained with
propidium iodide (PI, 36 µg/ml; Sigma, St. Louis, MO, USA) containing RNase
(10 µg/ml, Sigma) at 37 °C for 30 min. Cells were then analysed by CyAn™ ADP
flow cytometry (Beckman Coulter, Fullerton, CA, USA) according to the
manufacturer’s instructions, to determine the proportions of cells in
subG₁ and G₂/M phases. Apoptosis was measured as the
percentage of cells in the subG1 population, and data were analysed with ModFit
LT software, version 3.0 (Verity Software House, Topsham, ME, USA).

Isolated CD4⁺ T cells were stained with Cell Proliferation Dye eFluor 670 (eBioscience), following the manufacturer’s protocol. In brief, 2 × 10⁶ CD4⁺ T cells were washed three times with PBS and then resuspended in 500 μl of PBS at RT. Cell Proliferation Dye eFluor 670 was diluted with PBS to a final concentration of 10 μM. A total of 500 μl of the dye was added to 500 μl of the cell suspension while vortexing. Cells were then incubated at 37°C in the dark for 5 min. The reaction was stopped by adding 3 ml of cold FBS to the cell suspension and incubating on ice for 5 min. Cells were then spun down at 1200 rpm for 5 min at 4°C and washed three times with T cell media containing ≥10% FBS. Cells were then recounted, and 1 × 10⁶ cells per milliliter were activated with plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (4 μg/ml) for 3 d. Cells were then analyzed on day 3 by Cyan ADP flow cytometry (Beckman Coulter).