Muse cell cycler

HCT116 cells were treated with I3M, trypsinized, and washed in cold 1 × PBS. The cells were then incubated with MUSE^® annexin V and dead cell assay reagent (EMD Millipore, Billerica, MA) for 20 min and the early/late apoptotic cell population was measured by MUSE^® cell cycler (EMD Millipore, Billerica, MA, USA).

Cell viability and total cell numbers were measured by flow cytometric analysis. Briefly, MC3T3-E1 cells were seeded at 3 × 10³ cells/mL in six well plates for overnight and treated with the different concentrations (0–200 μg/mL) of FO for 1, 3, 5, and 7 days. DEX (100 nM) was used a positive control for MC3T3-E1 cell viability. After harvesting, the cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated with Muse^® cell count and viability kit (EMD Millipore, Billerica, MA, USA) for 5 min. Cell viability and total cell number were measured by Muse^® cell cycler (EMD Millipore).

To estimate viability, total viable cell count and dead cell percentage, flow cytometry analysis was carried out based on differential staining of viable and non-viable cells due to their different permeability to DNA binding dyes. B16F10 cells were plated at a density of 1 × 10⁴ cell/mL for 18 h and then treated with the indicated concentrations (0–800 μg/mL) of PS and PTS for 72 h. In brief, the cells were harvested and washed with ice-cold phosphate-buffered saline (PBS). Next, the cells were incubated with Muse® cell count and viability kit (EMD Millipore, Billerica, MA, USA) for 5 min and cell viability, total cell count and dead cell population were analyzed by using a Muse® cellcycler (EMD Millipore) according to the manufacturer’s instructions.