Dxflex ow cytometry system

Viability, apoptosis and necrosis were evaluated by Annexin V/Propidium Iodide (PI) staining. For this purpose, cells were seeded into six-well plates as 5 × 10 5 cell per well and incubated for overnight for attachment. Cells were then treated with rapamycin at respective doses for 48 and 72 hours. For staining, cells were detached by trypsinization, washed once with Dulbecco's phosphate buffered saline solution (DPBS, Thermo Fisher Scienti c, #14190144) and suspended in 1X ice cold Annexin V Binding Buffer (BioVision Inc., #1006) followed by labelling with 5 μL Annexin V-FITC reagent (Biolegend, #640906) and 1 µL PI solution (Thermo Fisher Scienti c, #P3566, diluted to 250 μg/mL in DPBS) by incubating for 10 minutes under dark conditions at room temperature. Cells were read with DxFLEX ow cytometry system (Beckman Coulter Inc.) and analysed by CytExpert software [25] (link). The experiment was done as triplicates and 2.5 × 10 4 cells per analysis were acquired at medium ow rate.

DNA content analysis was performed by using Cell Cycle Kit (Beckman Coulter Inc., #C03551). Cells were seeded as 5 × 10 5 cell/six-well plate and incubated overnight for attachment. After incubating with rapamycin at respective doses for 48 and 72 hours, cells were detached by tyripsinization, washed once with DPBS, xed with ice cold 70% ethanol by adding dropwise followed by incubation at 4°C for an hour. Tubes were stored at -20 °C overnight, and ethanol was removed by centrifuging cells at 400 x g for 5 minutes. Pellet was suspended in 500 µL Cell Cycle Kit reagent, and tubes were incubated at room temperature under dark for 30 minutes. DNA content was analyzed by DxFLEX ow cytometry system (Beckman Coulter Inc.) and analysed with ModFit LT 5.0 software (Verify Software House) [26] . The experiment was done as triplicates and 5 × 10 4 cells per test were acquired at medium ow rate. Analyses were performed