Caspase colorimetric assay kit

Saikosaponin (SSa; Nacalai Tesque Inc., Kyoto, Japan) was dissolved in DMSO (Sigma Chemical Co., St. Louis, MO) to form a 10-mM stock solution and stored at –20°C until use. Propidium iodide (PI), RNase A, Hoechst 33342, cytochalasin-B, Giemsa stain, and 4’,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. Caspase colorimetric assay kits were purchased from BioVision (Palo Alto, CA). Caspase-4 inhibitors, z-LEVD-fmk and Ac-LEVD-CHO, were obtained from Enzo Life Sciences (Plymouth Meeting, PA). The caspase-12 inhibitor, z-ATAD-fmk, was obtained from BioVision. z-DEVD-fmk (caspase-3 inhibitor), z-VDVAD-fmk (caspase-2 inhibitor), and z-IETD-fmk (caspase-8 inhibitor) were obtained from Calbiochem (La Jolla, CA). The annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Cyt c from equine heart was from Sigma-Aldrich^® (St. Louis, MO) and HA-NH₂ (10 kDa) from Creative PEGWorks (Winston-Salem, NC). The dithiobis(succinimidyl propionate) (DSP) cross linker was obtained from ProteoChem™ (Loves Park, IL). The cell lines A549 (human lung adenocarcinoma; ATCC^® CCL185 ™) and COS-7 (African green monkey kidney fibroblast) cells were from the American Type Culture Collection (Manassas, VA). CellTiter 96 aqueous non-radioactive cell proliferation assay was from Promega Corporation (Madison, WI). NucBlue^® Fixed Cell ReadyProbes^® Reagent (4′,6-diamidino-2-phenylindole, dihydrochloride, DAPI), fluorescein (FITC), Annexin Dead Cell Apoptosis Kit with Annexin V Alexa Fluor^® 488 and propidium iodide (PI) were purchased from Invitrogen (Carlsbad, CA). Caspase colorimetric assay kit was purchased from BioVision, Inc., San Francisco, CA and the caspase 9 substrate was from Sigma Aldrich (St. Louis, MO). All other chemicals (reagentor analytical grade) were purchased from various suppliers and used without further purification.

Caspase-3, caspase-8, and caspase-9 activities were tested according to the instructions of the caspase colorimetric assay kit (Biovision, CA, USA). PBMCs (1 × 10⁶) were lysed and centrifuged at 12000 g for 10 min. Thirty μg of protein from the extracts was incubated with 100 μL of enzyme-specific colorimetric substrates at 37°C for 2 h. The colorimetric release of p-nitroaniline from the substrate was measured using a 405 nm light wave. Absorbance was treated as a marker of activity.

We used a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan) for cell proliferation, a wound-healing assay for cell migration, BioCoat Matrigel invasion chambers (BD Biosciences, Bedford, MA, USA) for cell invasiveness, and the CytoSelect 48-Well Cell Adhesion Assay (Cell Biolabs, San Diego, CA, USA) for cell adhesion to extracellular matrix components. These assays were performed as previously described.² (link)^,40 (link) To evaluate total caspase activity, a Muse MultiCaspase Kit (Merck Millipore, Billerica, MA, USA) was used. The activities of caspase-3, -8, -9, and -12 were measured using the Caspase Colorimetric Assay Kit (BioVision, Milpitas, CA, USA). Mitochondrial membrane potential and cell-cycle distribution were assessed using a Muse MitoPotential Kit (Merck Millipore) and a Muse Cell Cycle Kit (Merck Millipore), respectively. ALDH, a surrogate marker of stem/progenitor cells, was estimated using the ALDEFLOUR fluorescent reagent system (STEMCELL Technologies, Vancouver, BC, Canada). ALDH-positive cells were determined using a FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA). The three-dimensional spheroid cultures were analyzed using PrimeSurface96U multiwell plates (Sumitomo Bakelite, Tokyo, Japan).

Caspase activities in rutin-treated SiHa cells were assessed by a caspase colorimetric assay kit (BioVision, Milpitas, CA, USA). Both untreated (control) and rutin-treated SiHa cancer cells were lysed in chilled buffer (cell lysis) and incubated for 10 min on ice. Further, centrifugation was performed at 10,000× g for 1 min to obtain supernatant, which was kept on ice for instant assay. Then, 50 μL of lysate was aliquoted into 96-well plates with 50 μL of reaction buffer containing 10 mm DTT. DEVD-pNA (5 μL) substrate was then added into each well and left to incubate for 1 h at 37 °C. Absorbance was recorded at 405 nm on a microtiter plate reader. Percent increase in Caspase-3 activity was determined by comparing the result of treated cells with untreated cells (level of uninduced control).

Caspase colorimetric assay kit (Biovision, USA) was used to evaluate caspase activity in LP-1 cells treated with plasma for 0.5 min, 1 min and 2 min. NAC was added to the cells at a final concentration of 10 mM followed by 2 min plasma treatment. After incubation for 6 h and 9 h, cells were resuspended in 50 μL cell lysis buffer and incubated on ice for 10 min. Then, the concentration of protein in the centrifuged supernatant was assayed. Samples with 150 μg protein in 50 μL cell lysis buffer were added to 96-well plates and cultured with 50 μL of 10 mM DTT and 5 μL of 4 mM substrate at 37°C for 1.5 h. The absorbance was read at 400 nm in a microtiter plate reader (Thermo Scientific).

A caspase colorimetric assay kit (Biovision, CA, USA) was used to measure the caspase -3 activity in treated cell line according to manufacturer’s instructions. The cells (10⁶/ml) were placed in a six-well plate for 24 h before treatment with various concentrations of FAA. After 24, 48 and 72 h, treated cells were collected into micro-centrifuge tubes and centrifuged at 1000 rpm for 5 min. Following two washes of pelleted cells with PBS, lysis buffer (50 μl) was added and mixed well. The cells were then incubated on ice for 10 min and subjected to centrifugation at 10,000 rpm for 1 min. Supernatants (50 μl) were transferred into wells of a 96-well plate; 50 ml of 2 × reaction buffer containing 0.5 μl DTT and 5 ml of caspases substrate was added to each sample. Samples were then incubated at 37 °C for 2 h in the dark. The cleavage of labelled substrate pNA into chromo-phore p-nitroanilide (pNA) was determined by measuring the absorbance (optical density, OD) at 405 nm using a FLUOstar Omega microplate reader (BMG Labtech, Ortenberg, Germany). The result of the induced group’s caspases activity was obtained by computing OD inducer/OD negative control with the background OD values from cell lysates and buffers subtracted [23 (link)].