Annexin 5 coupled fluorescein isothiocyanate fitc apoptosis detection kit 1

The relative percentage of apoptotic cells was assessed at 0, 30, and 40 hours after treatment with 0.2 μM KPT-185 using Annexin V-Coupled Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit-1 (BD Pharmingen, San Diego, CA) per the manufacturer’s protocol. Briefly, the cells cultured in a six-well plate were dissociated using trypsin-EDTA, then washed with phosphate-buffered saline and re-suspended in 100 μl of Annexin V-binding buffer at 1 × 10⁶ cells per milliliter. After adding 5 μl Annexin V-coupled FITC and propidium iodide (a probe to distinguish viable from nonviable cells) per 1 × 10⁵ cells, samples were mixed gently. After 15 minutes of incubation at ambient temperature in the dark, 400 μl of Annexin V-binding buffer was added. Analysis was performed using flow cytometry. Propidium iodide was excluded from viable cells which had intact membranes and permeated cells which were dead or had damaged membranes. Cells that stained positive for Annexin V-coupled FITC and negative for propidium iodide were identified as undergoing apoptosis. Cells that stained positive for both Annexin V-coupled FITC and propidium iodide were in the late stage of apoptosis, undergoing necrosis, or already dead. Cells that stained negative for both Annexin V-coupled FITC and propidium iodide were alive and not undergoing measurable apoptosis.