Apotox glo triplex assay kit

Cell viability, cytotoxicity and apoptosis events in the same well were measured using ApoTox-Glo™ Triplex Assay Kit (#G6320, Promega) with or without Z-VAD-FMK (#S7023, Selleckchem, TX, USA). GF-AFC substrate was used to detect live-cells and bis-AAF-R110 substrate was used simultaneously to measure dead-cell protease activity. Luciferin, a substrate of luciferase, was measured to quantify cleaved caspase-3/7, an important indicator of apoptosis. In the experiment with Z-VAD-FMK, cells were pre-treated for 1 h at its final concentration of 20 μM, prior to KPT-9274 treatment.

AlamarBlue® viability, total cell count, foci formation, suspension culture, and growth in 3D Matrigel culture were performed as previously described [40 (link), 47 (link)]. Biochemical assays, cell viability, apoptosis, and cytotoxicity were performed using ApoTox-Glo™ Triplex Assay Kit (G6320, Promega, China) as previously described [40 (link)]. Phosphatidylserine exposure and cell death, Live/Dead cell visualization, and IF analysis was performed as described previously [40 (link), 49 (link)]. Information of primary/secondary antibodies utilized are tabulated in SI 2C. Nuclei were stained in a mounting medium with DAPI (ab104193, Abcam). Combination index (CI) analysis was performed using the Chou-Talalay CI method [50 (link)].

Cell migration and invasion assays were performed using BD BioCoat Matrigel invasion chambers (BD Biosciences, Bedford, MA) according to the manufacturer's instructions and as previously described [32 (link)]. The colony scattering assay was performed as previously described [62 (link)]. ApoTox-Glo^™ Triplex Assay Kit from Promega was performed according to the macufacturer's instructions and as previously described [63 ]. Cell functional assays, including an AlamarBlue® viability assay, anchorage-independent growth (soft agar colony formation and foci formation), 2D and 3D morphogenesis, and wound-healing assay were performed as previously described [32 (link)]. The collagen I adhesion assay was performed on Collagen I substrate coated plate following manufacturer's instructions (Invitrogen, Singapore). The endothelial cell adhesion and endothelial trans-migration assays were performed as previously described [62 (link)].

PC3 cells were seeded at 8000 ​cells per well in black flat bottom Greiner CELLSTAR 96 well plates, (100 ​μL media per well) and incubated for 24 ​h at 37 ​°C under an atmosphere of 5% CO₂/95% Air. After 24 ​h elapsed, the cells were exposed to Dido (10, 30 and 50 ​μM) or vehicle (1% v/v) and incubated for a further 6 ​h. Caspase 3/7 cleavage was then quantified using the ApoTox-Glo™ Triplex Assay kit (Promega) as per manufacturer's instructions.

The assay was carried out using Apotox-Glo™ Triplex assay kit (Promega, USA), following manufacturer’s protocol. Briefly, 7 × 10³ cells/well/100 μL culture media were seeded at 37 °C in a 96-well white plate for 24 h. The cells were then treated with MP-HX and MP-EA for 24 h and 48 h. The concentration of the extracts used in the assay were at ~IC₅₀ values for HCT116, HCC1937, HepG2 and MDA-MB-231 cells, where MP-HX concentrations were 70 μg/mL, 90 μg/mL, 75 μg/mL, and 45 μg/mL, respectively, while MP-EA concentrations were 75 μg/mL, 90 μg/mL, 130 μg/mL and 90 μg/mL, respectively. After adding the Caspase-Glo® 3/7 reagent, the cells was incubated for 30 min at room temperature and the luminescence reading was measured using Tecan Infinite® M1000 Pro multimode reader.

Apoptosis, viability and cytotoxicity were assessed after heat shock, with the ApoTox-Glo Triplex Assay kit (Promega, cat. no. G6320) according to previously described methods with minor modifications^{22 (link)}. To expose cells to heat shock stress, 10,000 fibroblasts per well were first grown at 37 °C in 96-well ELISA microplates and allowed to attach overnight. The following day the growth medium was exchanged with pre-warmed medium and the cells were moved into a 43 °C incubator for 2 h of heat stress exposure. Cells were then returned to 37 °C to recover for various time points. To provide a positive control for the induction of apoptosis, 2.5 µM staurosporine was added to the growth medium for 6 h. To provide a positive control for cytotoxicity, 70 µM digitonin was added to the culture medium for 15 min. Fluorescence (viability (400Ex/505Em) and cytotoxicity (485Ex/520Em)) and luminescence were measured on a FLUOstar Optima microplate reader (BMG Labtech).