Cell Cycle Staining/Analysis Sample Clauses

Cell Cycle Staining/Analysis. Propidium iodide (PI) staining of nuclear chromatin was used to quantify the number of cells from a given population in each respective phase of the cell cycle. 5x104 cells were seeded in 12 well plates usually in triplicate. After treatment cells were cultured for 48/72h as indicated and harvested with TrypLE™. Cells were transferred to FACs tubes containing PBS+2% FCS to neutralise the TrypLE™ and centrifuged at 1000xg for 5 mins. Media was removed by flicking and cells were resuspended by running individual FACS tubes across a draining board grate. Tubes were then individually vortexed as 400μl of 70% ethanol in ddH2O was added to prevent cell clumping. Cell fixing was for 30 mins at RT or for <1 week at 4⁰C. For staining cells were once again centrifuged at 1000xg for 5 mins and fixing buffer was flicked off. Cells were resuspended as described. Staining solution was 0.05% Triton-X in PBS + 50μg/ml PI+100μglml RNase A (Both Sigma-▇▇▇▇▇▇▇). 500μl of staining solution was added to each tube and incubated in the dark at 37⁰C for 45 mins. In this time RNaseA digests the cells’ RNA content which is necessary as PI binds to both DNA and RNA and intact RNA remaining in the sample will invalidate the result. Following staining cells are centrifuged once more and remaining staining buffer flicked off and cells resuspended. Cells are then acquired using the FACS canto II. Data was analysed using the 1D cell cycle analysis feature in FlowJo 7.6.4.