Modfit 3

Cell cycle analyses were performed with the Muse^TM Cell Analyzer according to the Muse™ Cell Cycle Kit user's guide, after RPE cells were cultured in whole culture medium and 100 µg/ml PSPA culture medium for 1–3 days. For each sample, 20,000 events were recorded. The results were analyzed using ModFit 3.2 (Verity Software House, Topsham, ME, USA).

In order to confirm the MTS results, the cell cycle of DOK cells and OSCC-BD cells was analyzed using a flow cytometer. DOK cells and OSCC-BD cells were subcultured for 48 h, and dispersed by trypsinization and suspended in PBS, then the cells were stained with propidium iodide and measured using a flow cytometer (LSR II, BD Biosciences, USA). The cell cycle distribution was analyzed by computer software (ModFit3.2, Verity Software House, USA). The procedure was performed according to the manufacturer’s protocol.

Cell cycle distribution was analyzed as previously described [25 (link)]. Cells were seeded in 6-well plates (2.0 × 10⁵ cells/well), allowed to adhere overnight, and at T0 were exposed to PTX (5–50 nM) and/or BRP (0.1–3 μM). The vehicle control was treated with 0.1% (v/v) DMSO. Just before drug exposure at T0, and after 17, 48, and 72 h of drug exposure, adherent cells were harvested, counted by Coulter counter (Hialeah, FL) and stained with PI/RNase staining buffer. At least 10,000 events from triplicate samples were collected using a FACSCalibur flow cytometer (Becton–Dickinson, Mansfield, MA), and cell cycle distributions were analyzed using ModFit 3.2 (Verity Software House, Topsham, ME). The fitted ModFit curves were smooth, and debris and aggregates were < 5%. Cells in the G0/G1- and G2/M-phases were identified based upon DNA content of 2 N and 4 N, and the intensity ratio for G2/G1 was 1.8–1.9. DNA content for cells in S-phase was between 2 N to 4 N; content > 4 N was defined as polyploid. The sub-G1 population was minimal and excluded from quantification.

Analysis of the PBMC cell cycle distribution was performed after 72 h of incubation with or without different concentrations of CB[n] (0.5, 0.3, and 0.1 mM). After cultivation, the PBMCs were washed from the medium with phosphate-buffered saline (Biolot, St Petersburg, Russia). Then, cells were fixed overnight at −20 °C in 70% ethanol and stained with propidium iodide (100 µg/mL) in the presence of RNAse A (100 µg/mL) for 30 min at 37 °C. Cell cycle analysis was conducted by evaluating DNA histograms. Samples were analyzed on a FACSCanto II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and ModFit 3.2 software (Verity Software House, Topsham, ME, USA). The relative amounts of cells with diploid (cells in G₀/G₁ phases of the cell cycle) and hyperdiploid (cells in S and G₂/M phases of the cell cycle) DNA sets were determined. Cells with fragmented DNA formed a characteristic hypodiploid peak.