Detecting Apoptosis via Nuclear Condensation

To detect nuclear condensation indicative of apoptosis, NucBlue Live Cell Stain (Hoechst 33342) was used (Invitrogen, Carlsbad, CA). Hoechst staining was performed as described previously [16 (link), 20 (link)]. For A2780 and A2780-CDDP, cells were treated with 12.5 μM BT and 4 nM paclitaxel either alone or in combination for 48 hours. Similarly, IGROV-1 and IGROV-1-CDDP cells were treated with 50 μM BT or 4 nM paclitaxel for 48 hours either alone or in combination. Following treatment, cells were washed stained with Hoechst stain (2 drops/mL of media) for 15 min. at 25°C and observed under a fluorescent microscope. Representative images were taken with an inverted microscope (Olympus H4-100, CCD camera) and 20× objective. Duplicate wells were set up for each treatment condition. Each experiment was repeated three times. After morphological assessment by nuclear staining, the extent of apoptosis was quantified using the TUNEL assay.