Cell counter r1

4T1 cells with 2N DNA content (G₀–G₁) were isolated following the treatment of DMSO or 1 μM FiVe1 for 72 h. Cells were stained with 5 μg/mL Hoechst 33342 in monolayer culture for 2 h in a 37°C cell culture incubator. Cells were then dissociated with TrypLE (Gibco) and passed through a 35-μm cell strainer (Corning). The concentration of viable cells was determined by staining with trypan blue and counting on a Cell Counter R1 (Olympus). Cells were sorted using a MoFlo Astrios Cell Sorter (Beckman Coulter) at the MD Anderson Flow Cytometry & Cellular Imaging Facility. The DMSO-treated cells, with a normal distribution of cells in the cell cycle, were used to gate for the 2N population. The cells in the 2N population were isolated for subsequent experiments.

Cells in monolayer culture were disassociated with TrypLE (Gibco) and passed through a 35-μm cell strainer (Corning), resulting in single-cell suspensions. Cells were then stained with trypan blue and counted using a Cell Counter R1 (Olympus) to determine the concentration of viable cells. We plated 500 viable cells per well in an ultra-low attachment 96-well plate in 100 μL of the mammosphere media (MEGM media with 1% methylcellulose containing 10 ng/mL EGF, 20 ng/mL FGF, and 4 μg/mL heparin) as described.^{34 (link)} Mammosphere media was replenished every 3 days. Cells were either pretreated in monolayer cultures or treated during the mammosphere assay as indicated. After 12 days of treatment, each well was imaged using a Cytation3 Plate Reader and Imager (BioTek) at 4x magnification and stitched together into a tile using the BioTek Gen5 program. Spheres larger than 70 μm in diameter were considered mammospheres and counted manually.^{34 (link)}