Propidium iodide

For determination of cell density, cells were plated in 96-well plates at 5 × 10³ cells/well. At 24 h, cells were treated with specified drugs for 48 h (or otherwise indicated). After treatment, media were removed, and plates were stained with a solution containing 0.5% crystal violet and 25% methanol, rinsed, dried overnight, and re-suspended in citrate buffer (0.1 M sodium citrate in 50% ethanol). Intensity of staining, assessed at 570 nm and quantified using a V_Max kinetic microplate reader (Molecular Devices Corp., Menlo Park, CA), is directly proportional to cell number [50 (link), 51 (link)]. For assessing cell viability and cell death (apoptosis and necrosis), cells were treated for 48 h, and stained with an Annexin V-fluorescein isothiocyanate and propidium iodide, respectively (Trevigen, Gaithersburg, MD).

Cells were fixed in 70% ethanol and stained in PBS containing 0.1% Triton X-100, 50 μg/mL RNase, and 50 μg/mL propidium iodide. Samples were analyzed by, flow cytometry and the hypodiploid peak constituted the apoptotic population in this analysis. DNA content was measured on a FACSort flow cytometer (Becton Dickinson, Franklin Lakes, NJ), and data were analyzed using ModFit software (Verity Software House, Topsham, ME). At least 1x10⁶ cells were analyzed per sample. For Annexin V Labeling, samples were stained with fluorescein-labeled Annexin V and propidium iodide (Trevigen) according to the manufacturer’s protocol. The two Annexin V positive quadrants of the analysis were taken as the apoptotic fraction. Anoikis Assays were carried out as described previously [39 (link)]. To perform the analysis of cell surface markers (CD44+/CD24-, CD49+/CD24-), MCF10A cells were detached with trypsin, washed in blocking buffer (PBS containing 3% FBS), then stained with anti-CD24-PE (BD Biosciences, San Diego, CA, USA) and anti-CD44-PE-Cy5 (BD Biosciences) or anti-CD49-FITC (BD Biosciences) using 1 ml of antibody per 10⁶ cells, and incubated at room temperature for 1 hr. Following incubation, cells were washed twice with 1 ml PBS. Cells were re-suspended in 1 ml PBS and then analyzed.