at room temperature. The luminescence intensity was measured using lumin ometer. Ganetespib cancer Cells treated with vehicle were considered as control against which treated cells were compared. Experiments were performed in triplicate. Inhibitors,Modulators,Libraries Data was expressed as mean SD of triplicate experiments. In addition to homogenous caspase 3 7 assessment, we also analyzed expression of effector caspases, e. g, caspase 3 and caspase 7 via immunoblotting using specific antibodies against caspase 3 and 7. Morphological studies to detect apoptosis To detect nuclear condensation indicative of apoptosis, NucBlue Live Cell Stain was used. Hoechst 33342 is a cell permeant nuclear counter stain that emits blue fluorescence when bound to DNA. It is excited by UV light and emits blue fluorescence at 460 nm when bound to DNA.

To detect apoptotic specific nuclear changes, cells were seeded into Inhibitors,Modulators,Libraries 12 well plate and treated with sub cytotoxic BT at concentrations of 25 uM, 50 uM or 100 uM for 6 or 24 hrs. Following treatment, cells were washed with PBS twice and fresh media containing Hoechst was Inhibitors,Modulators,Libraries added. Cells were incubated 15 min. at 25 C and observed under fluores cent microscope. Representative images were taken with an inverted microscope and 20 objective. After morphological assessment by nuclear staining, extent of apoptosis was quantified using the TUNEL assay. TUNEL assay DNA fragmentation was detected using the TiterTACS 2 TdT In Situ Colorimetric Apoptosis Detection Kit following the manufacturers instructions. Briefly, cells were seeded at a density of 3 104 cells well, into 96 well flat bottom plates and incu bated for overnight.

Cells were treated with Inhibitors,Modulators,Libraries BT as described previously. Following treatment, cells were washed and fixed followed by addition of labeled nucleotides and subsequent detection with HRP HRP Inhibitors,Modulators,Libraries substrate system. The absorbance was measured at 450 nm using a microplate reader, Multiskan. Mitochondrial transmembrane depolarization potential assay Mitochondrial transmembrane depolarization potential was determined by flow cytometry using Rhodamine 123. Ovarian cancer cells were seeded in a 100 mm2 culture dishes and treated with 50 uM or 100 uM BT for 6 or 24 hrs. Following treatment, cells were harvested by trypsinization, washed with PBS, and resuspended in fresh DMEM medium containing rhodamine 123 at a concentration of 0. 5 mg mL, and incubated for 30 min. at 37 C.

The cells were washed twice with DPBS, re suspended in DPBS and analyzed by flow cytometry. Data was acquired on a BD Accuri C6 flow cytometer and analyzed. Twenty selleckchem Gemcitabine thousand cells were analyzed for each sample. Appropriate gating was used to select standardized cell population. Cell cycle analysis Cell cycle analysis was carried out by flow cytometry. Cells were seeded into 100 mm2 tissue culture dishes and treated with 50 uM BT for 24 hrs.