Dna prep stain

Indicated HCC cells were harvested 72 h after indicated treatment. Then the cells were fixed in 75% ethanol overnight at –20°C after washed with PBS. Then the cells were washed with PBS for twice and applied with DNA Prep Stain (Beckman Coulter, USA) and RNase for 30 min. Next, the cell cycle analysis was performed by Flow Cytometry System with FACSDiva software. The data were analyzed by ModFit LT 3.2 software (Verity Software House, USA) and the cell cycle distribution was described as the percentage of cells in G1, S, and G2 populations.

After 48 h transfection, UM-UC-3 and T24 cells were washed with PBS and fixed in 75% ethanol at ?20°C. After 24 h fixation, the cells were washed with PBS and treated with DNA Prep Stain (Beckman Coulter, Fullerton, CA) for 30 min. Cell cycle analysis was conducted by BD LSRII Flow Cytometry System with FACSDiva software (BD Bioscience, Franklin Lakes, USA). The cell cycle distribution was illustrated as the percentage of cells in G1, S, and G2 populations and data was evaluated by ModFit LT software package.

PMNL (1 × 10⁶/mL) were treated with dabigatran or rivaroxaban at a final concentration of 10 μM for 120 min at 37 °C, followed in succession by a 10 min exposure of the cells to propidium iodide (DNA prepstain, Beckman Coulter, Miami, FL, USA, 50 μg/mL) at room temperature and flow cytometric detection of uptake of propidium iodide as a marker of cell membrane damage expressed as % viable cells.

This was measured after a 15 min incubation period following the addition of DMSO (resting control) or dolutegravir (10 μg/mL) to neutrophils (1 × 10⁶ /mL) using a flow cytometric propidium iodide-based dye exclusion assay. The cells were incubated for 10 min with propidium iodide (50 μg/mL, DNA Prep-Stain, Beckman Coulter, Miami, FL, USA), and cell viability was assessed using a FC500 flow cytometer (Beckman Coulter, Miami, FL, USA). The results are expressed as the percentages of viable cells. The analysis was performed using FlowJo version 10.8.1 for Windows (BD BioSciences, Franklin Lakes, NJ, USA). Bisector gates were applied to distinguish between propidium iodide negative (viable cells) and propidium iodide positive populations (apoptotic cells) in the control. The same gates were applied to all subsequent samples.

For proliferation assay, cells were seeded in 35 mm Petri dish at a concentration of 5 × 10⁴ cells/dish in a complete medium. Cells cultured on ground or on RPM for 24, 48 and 72 h were counted with a particle count and size analyzer (Beckman-Coulter, Inc., Fullerton, CA, USA) and by a Thoma hemocytometer. Three independent experiments in duplicate were performed. For cell cycle analysis, cell clumps were collected and centrifuged and pellets were trypsinized and washed twice with PBS (Phosphate Buffered Saline, Sigma–Aldrich, St. Louis, MO, USA). Adherent and ground control cells were trypsinized and washed twice with PBS. Cells were fixed with 70% ethanol at 4 °C for 24 h and stained with DNA PREP Stain (Beckman Coulter, Fullerton, USA) at 4 °C overnight. Stained cells were measured by flow cytometry. Cell cycle analysis was performed three times.