Caspase 3 assay kit

Caspase-3 activity was determined by using a caspase-3 assay kit (PharMingen, San Diego, CA) according to the manufacturer's instructions. The cells (1×105) were homogenized in a lysis buffer containing 0.1% CHAPS, 50 mM HEPES (pH 8.0), 12.5 mM NaCl, 0.1 mM EDTA, and 5 mM DTT freshly added. The whole cell lysates (40 mg) were incubated with 20 mM of colorimetric substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) in a reaction buffer (BioVision) containing 5 mM DTT at 37°C for 1 h. pNA was released from the substrate upon cleavage by DEVDase. The yellow color produced by free pNA was monitored by a spectrophotometer at 405 nm. The amount of yellow color produced upon cleavage was proportional to the amount of DEVDase activity.

Caspase-3-like activity was assayed according to the manufacturer’s guidelines (caspase-3 assay kit, BD Pharmingen, USA). Briefly, a 1 ml aliquot of 24 h grown culture was washed twice with phosphate buffered saline (PBS) (10 mM, pH 7.5) and resuspended in saline (0.85%). The cell suspension was centrifuged at 12,500×g for 10 min. The pellet was resuspended in 100 µl of sodium phosphate buffer (10 mM, pH 7.5), mixed with 1 ml cell lysis buffer {Tris-HCl (10 mM), sodium phosphate buffer (10 mM, pH 7.5), NaCl (130 mM), triton X-100 (1%) and sodium pyrophosphate (10 mM)} and kept at 4°C for 4 h for lysis. The cell lysate was then centrifuged at 12,500×g for 15 min and an aliquot (50 µl) of the above supernatant was used for caspase-3 assay using synthetic fluorogenic substrate Ac-DEVD-AMC (BD Pharmingen, USA) as described earlier [2] (link).

Caspase-3 activities were measured using a Caspase-3 assay kit (BD Biosciences, USA) based on hydrolysis of the substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA), resulting in release of the p-nitroaniline (pNA) moiety. Released pNA is detected at 405 nm. Comparison of pNA absorbances from the sample and control allows determination of the fold increase in caspase-3 activity (relative caspase-3 activity is expressed in arbitrary units).

Caspase-3 activity was measured using a caspase-3 assay kit (BD Pharmingen, San Diego, USA) according to the manufacturer's instructions. Cells were treated with IFNβ (0–72 h), washed twice with cold PBS, lysed on ice in 1 ml lysis buffer (Cytofix/Cytoperm™ Fixation and Permeabilization Solution) and further incubated for 20 min on ice. Cell lysates were then centrifuged at 1,000 rpm for 5 min. The pellet was washed twice with Perm/Wash™ Buffer and labeling performed by adding 100 μl of washing buffer containing 20 μl of antibody (BD Pharmingen). The samples were incubated for further 30 min at room temperature, washed in washing buffer and then analyzed by flow cytometry. A negative control sample incubated with FITC-immunoglobulin G was run in parallel. Three independent experiments were performed for each assay.

Cell viability was measured by trypan blue (Sigma-Aldrich, Poznan, Poland) exclusion test and the cytostatic index (IC₅₀) was determined. The number of viable cells after 4 days of exposure to CIF or EGCG or genistein or DAC was given as a percentage of viable cells in the vehicle control. The IC₅₀ value represents the growth inhibitory concentration at which the compound causes 50% decrease in the number of viable cells compared with vehicle control after 4 days incubation. The number of dead cells that took up trypan blue was specified as the percentage of the total cell number. Cells were counted using Countess automated cell counter (Invitrogen, Life Technologies, Warsaw, Poland). The number of viable, necrotic, early, and late apoptotic cells was determined after 4 days compound exposure by flow cytometry analysis using annexin V/propidium iodide (PI) (FITC Annexin V Apoptosis Detection Kit, BD Pharmingen, Warsaw, Poland) staining according to the manufacturer’s protocol. Caspase-3 assay (Caspase-3 Assay Kit, BD Pharmingen, Warsaw, Poland) was performed to estimate its activity as a marker of the early stage of caspase-dependent apoptotic pathway in MDA-MB-231 cells (in MCF7 cells functional deletion in CASP3 gene) [51 (link)].