Annexin 5 pi double staining kit

Apoptosis was evaluated using an Annexin V/PI double staining kit (Merck, Germany) according to the manufacturer's instructions. Briefly, single cell suspensions were incubated with Annexin V solution and stained by propidium iodide (PI) solution for 10 min at room temperature. Then, the cells were harvested and analyzed with a Becton Dickinson FACS CantoII Flow Cytometer (Beckman Coulter, USA).
To analyze DNA content and cell cycle by flow cytometry, the cells were harvested, washed twice with PBS, and fixed with 70% ethanol. Immediately before flow cytometric analysis, the cells were washed twice in PBS and then stained for DNA content using 0.5 ml of 400 µl/ml PI (Sigma, USA) and 100 μg/ml RNAase A (Sigma, USA) in PBS and 38 mM sodium citrate pH 7.4. Data were again collected on a Becton Dickinson FACS Canto II Flow Cytometer.
Cell culture and L-leucine/L-glutamine uptake assays TE-1 cells were cultured in RPMI-1640 medium containing 10% v/v FBS, penicillin-streptomycin solution, and 1 mM sodium pyruvate and maintained at 37°C in 5% CO 2 [ 3 H].-L-leucine and [ 3 H]-L-glutamine uptake assays were performed as described previously [18, 19] .