Apoptosis was determined using cleaved caspase 3 IF as previously described [12 (link)]. Briefly 40,000 cells were seeded in 12 well plates on glass coverslips in triplicate. 24 hours later cells were treated with chemotherapeutic agents with or without A69 for 24 hours. After treatment, cells were fixed in 3.7% formaldehyde for 15 minutes and permeabilized in 0.3% Triton X-100 for 15 minutes. Antibodies were diluted in blocking buffer that consisted of 0.2% non-fat dry milk, 2% Bovine Serum Albumin and 0.3% Triton X-100 in phosphate buffered saline (PBS). Primary anti-cleaved caspase 3 rabbit monoclonal antibody (1:400, Cell Signaling Technology, Danvers, MA) was applied to cells for 1 hour at 37°C. Following washes in PBS, samples were incubated in goat-anti-rabbit Alexa Fluor 488 (1:1000, Life Technologies, Carlsbad, CA) and Alexa 555 conjugated Phalloidin (1:200, Life Technologies) to visualize F-actin. Slides were mounted with Fluoromount G with Hoescht (Sigma) to label cell nuclei. The percentage of positive cells was determined for each assay with at least 150 cells being counted per condition using the counting feature on an Evos Xl core microscope. Each assay was run in triplicate and repeated three times. Representative images were taken on Zeiss Axio A1 Microscope with an AxioCam MRc digital camera.
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