Lsr fortessa b

Cells were treated with various concentrations of DFO or DFX for 48 hours or 120 hours, or with staurosporine (1 μM) for 18 hours as a positive control for apoptosis. The non-adherent cell fraction was collected from media, while the adhered cell fraction was harvested using trypsin. Cell fractions were re-combined, washed with FACS buffer, re-suspended in 100 μL of assay buffer, and stained for the Annexin V apoptosis marker and 7AAD non-specific cell death marker using the PE Annexin V apoptosis detection kit (Biolegend, San Diego, CA) per manufacturer’s instructions. Sample acquisition was performed using a BD FACS Caliber or LSR Fortessa B (BD Biosciences), with at least 10,000 events recorded per sample using Cell Quest or Diva software.

Cells were treated for 48 hours with 125 μM DFO and harvested using Trypsin (0.25%, Corning). Harvested cells were washed and fixed in 2 mL 70% ethanol (VWR, Radnor, PA) for 1 hour at 4°C. Fixed cells were washed in 2 mL FACS buffer (PBS, 5% FBS, and 0.01% sodium azide) and stained in 500 μL propidium iodide solution [0.05 mg/mL (Sigma-Aldrich, St. Louis, MO), 0.1% sodium citrate (Alfa Aesar, Heysham, England), 0.02 mg/mL RNAse (Thermo Fisher Scientific), 0.2% NP40 (Sigma-Aldrich), 1N HCl, DI water] for 1 hour at 4°C. Sample acquisition was performed using the LSR Fortessa B (BD Biosciences) with at least 10,000 events recorded per sample using the Diva software. Cell cycle analysis was performed using the ModFit LT 4.0 software (Verity Software House, Topsham, ME) in which cell counts in G1, S and G2 phases were measured.

Cells were harvested using Trypsin (0.25%, Corning, Tweksbury, MA), washed with Phosphate Buffered Saline (PBS) and incubated in 500 μL Phen Green SK Diacetate (5 μM, Thermo Fisher Scientific, Grand Island, NY) for 30 minutes at 37°C. Stained cells were washed once and resuspended in 200 μL PBS, to which 5 μL of 1:10 diluted Live-Dead Yellow (LDY) stain (Thermo Fisher Scientific) was added. The mixture was incubated for 10 minutes at room temperature in the dark. Sample acquisition was performed using the LSR Fortessa B flow cytometer (BD Biosciences, San Jose, CA) with at least 10,000 events recorded per sample using the Diva software. The Mean Fluorescence Intensity (MFI) of Phen Green staining was calculated for viable cells (LDY negative) using the WinList 9.0.1 software (Verity Software House, Topsham, ME).