Annexin 5 fluorescein isothiocyanate

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).

For determination of cell number, 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 resuspended in citrate buffer (0.1 M sodium citrate in 50% ethanol). Intensity of staining, assessed at 570 nm and quantified using a VMax kinetic microplate reader (Molecular Devices Corp., Menlo Park, CA), is directly proportional to cell number [20 (link)]. For apoptosis and necrosis, cells were treated for 48 h, and stained with an Annexin V-fluorescein isothiocyanate and propidium iodide, respectively (Trevigen, Gaithersburg, MD). Autophagy was detected by detecting SQSTM1/p62 and LC3II proteins by Western blotting. For the reactive species assay, cellular levels of total reactive species (RS; kit measures both oxygen and nitrogen species) were determined using the Total ROS detection kit (Enzo Lifesciences) and measured by Flow Cytometry and Cell Sorting Shared Resources.