Caspase glo 9 assay

We incubated 1.0 × 10⁴ cells/well with the peptides–NPs at the concentration of 1 µM for 6 h. The apoptotic process onset was evaluated by the Caspase-Glo^® 9 assay (Promega), according to the manufacturer’s instructions. After 30 min, the luminescence was measured using a Synergy HT microplate reader (Biotek).

Promega Caspase-Glo 9 assay (Promega, Madison, WI, USA) was employed to evaluate the caspase-9 activity. T47-D cells were seeded in a 96 multiwell plate at a density of 4 × 10⁴ cells/mL. After 48 h, cells were treated with the positive control, DOX, at 10 μM and with 10 and 20 μM of 3g, during 48 h. The assay was performed at the end of treatment, following the instructions provided by the manufacturer. Data are representative of at least two experiments (n = 4 in each).

The activity of caspase Dronc was measured using Caspase-Glo 9 Assay (Promega, United States). Male flies, 7- or 30-day-old, were decapitated at ZT16. Heads were collected and then lysed for 30 min at 4 °C with an HBSS buffer, pH 7.9, supplemented with Na₂EDTA (0.1 mM, POCH, Poland), EGTA (0.1 mM, Sigma, Germany), and 2% NP-40. The amount of the extraction buffer used for cell lysis followed a 1:1 ratio of the number of heads and the volume of the extraction buffer (1 μl per head). Then, the samples were centrifuged at 13,200xg for 1 h at 4°C. The supernatant was collected and protein content was estimated by Nanodrop 2000. Protein solutions were then mixed with the Caspase-Glo^® 9 reagent at a 1:1 ratio, according to the manufacturer’s protocol, to individual wells in a 96-well plate. After 35 min of incubation at room temperature in darkness, the Relative Light Unit (RLU) of each sample was measured using the end-point, luminescence (LUM) read mode function of a plate reader (SpectraMax iD3, Molecular Devices, San Jose, CA, United States). Whole-head homogenates were used for this experiment with at least three repetitions per genotype and at least 40 fly heads for each replicate. This headcount was initially calibrated with the total protein content that has been tested to detect caspase activity using cells from in vitro culture.

Caspase 3/7, caspase 8, and caspase 9 activities in D. cinnabari-treated H400 cells were measured using caspase-Glo 3/7, caspase-Glo 8, and caspase-Glo 9 assay kits from Promega according to manufacturer protocol. Briefly, 100 μL of 1 × 10⁵ cells/mL of H400 cells was seeded in a 96-well white plate and kept under 5% CO₂ at 37°C for 24 hours. Cells were then treated with DC extract, fractions DCc and DCd, and subfractions DCc15 and DCd16 of D. cinnabari using IC₅₀ concentration for 24 hours. Untreated cells were used as negative controls. MG132 inhibitor was added for caspase 8 and caspase 9 assays. Briefly, caspase-Glo buffer was thawed and equilibrated to room temperature prior to use. This buffer was then used to dissolve lyophilized caspase-Glo substrate and the mixture was vortexed to obtain a homogenous solution. Prior to the addition of caspase-Glo reagent, culture plate was equilibrated to room temperature. Then, 100 μL of the reagent was added to each well and placed on orbital shaker for 30 seconds. The plate was then further incubated at room temperature for 2 hours in dark. The luminescence signal was then measured using Tecan Infinite M200 Pro ELISA plate reader (Männedorf, Switzerland).

Caspase-3/7, caspase-8 and caspase-9 activity was assayed by measuring the light intensity using an assay kit (Caspase-Glo® 3/7Assay, Caspase-Glo® 8 Assay and Caspase-Glo® 9 Assay, Promega) with some modification. Briefly, ZR751 cells (1 × 10⁵ cells/well) were treated with different concentrations (5–40 μg/ml) of test samples for 24 h. After incubation, cell pellets were lysed in lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride and protease inhibitor cocktail). Equal amount of protein extract cell lysates were loaded in opaque white 96-well plates and 40 μl of caspase-3/7 or caspase-8 or caspase-9 reaction buffer was added and incubated at room temperature for 1 h before measurement.

Cells (1250 cells/well) were cultured in 386-well plates and incubated with DMSO, 100 or 200 nM S63845 for 6 h. The activity of caspase 3/7, 8, and 9 was assessed using the Caspase-Glo 3/7 Assay, Caspase-Glo 8 Assay, and Caspase-Glo 9 Assay (Promega, Madison, WI), respectively, according to the manufacturer’s recommendations. The measured value was corrected by the cell number using the CellTiter-Glo 2.0 Luminescent Cell Viability Assay.
The FITC Annexin V Apoptosis Detection Kit 1 (BD biosciences, San Jose, CA) was used to detect apoptosis. One million cells were suspended in 2 ml of culture medium and incubated at 37.0 °C in 5% CO₂ for 4 h after treatment with DMSO or 100 nM S63845 in the presence or absence of 50 µM Z-VAD-FMK. According to the manufacturer’s protocol, the cells were stained with FITC-conjugated annexin V and PI, followed by flow cytometric analysis using BD FACSMelody™ (BD biosciences, San Jose, CA). The percentage of the FITC annexin V-positive population was defined as the percentage of apoptosis.