H4 100

To detect nuclear condensation indicative of apoptosis, NucBlue Live Cell Stain (Hoechst 33342) was used (Invitrogen, Carlsbad, CA). Hoechst 33342 is a cell-permeant nuclear counter-stain that emits blue fluorescence when bound to DNA [15 ]. It is excited by UV light and emits blue fluorescence at 460 nm when bound to DNA. To detect apoptotic specific nuclear changes, cells (1 × 10⁵ cells) were seeded into 12-well plate and treated with sub-cytotoxic BT at concentrations of 25 μM, 50 μM or 100 μM for 6 or 24 hrs. Following treatment, cells were washed with PBS twice and fresh media containing Hoechst (2 drops/mL of media) was added. Cells were incubated 15 min. at 25°C and observed under fluorescent microscope. Representative images were taken with an inverted microscope (Olympus H4-100, CCD camera) and 20× objective. After morphological assessment by nuclear staining, extent of apoptosis was quantified using the TUNEL assay (described below).

Nuclear condensation indicative of apoptosis was assessed using NucBlue Live Cell Stain Hoechst 33342 (Invitrogen, Carlsbad, CA). This qualitative test was performed as described previously [30 (link), 32 (link)]. In brief, cells (1 × 10⁵ cells) were seeded in 60 mm² culture dishes and incubated for 4 days. At the end of the stipulated time, cells were washed and stained with Hoechst stain (2 drops/mL of media) for 15 minutes 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. Further quantification of apoptosis was done using Muse’s Annexin V & Dead Cell Assay Kit (MCH100105, MilliporeSigma, CA, USA) following the manufacturer’s instructions. Briefly, cells were incubated with Muse Annexin V & Dead Cell reagent at room temperature in the dark and analyzed using Muse® Cell Analyzer (Merck Millipore, Burlington, MA, USA). The Muse Software Module performs calculations automatically.

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.