Enzchek caspase 3 assay kit

Apoptosis, or programmed cell death, plays a critical role in assessing the potency of drug delivery systems and is a major pathway for cellular death. Caspase-3 activity was measured using an EnzChek^™ Caspase-3 Assay Kit (Molecular Probes, Eugene, OR, USA) as per the manufacturer’s specifications. Briefly, A549 cells were seeded at a density of 1 x 10⁶ cells/TC dish of a 100-mm diameter dish (Thermo Scientific, Rochester, NY, USA) and treated with PFD or PFD–D-Lip (0.25 mg/mL) for 6 h followed by harvesting and washing of cell pellets. Cell lysis was carried out using 1X cell lysis buffer while subjected to a freeze–thaw cycle, followed by centrifugation. Supernatants obtained were transferred to a 96-well plate to which 50 µL of 2X substrate working solution (10 mM Z-DEVD-AMC substrate + 2X reaction buffer) was added and incubated for 20 min. Then fluorescence was measured at excitation/emission 342/441 nm [42 (link)].

A 20-mg portion of tissue was homogenized in lysis buffer containing Tris HCl 200 mM pH 7.5, NaCl 2 M, EDTA 20 mM, Triton X-100 0.2% and centrifuged for 5 min at 5000× g. Fifty microliters of the supernatants were incubated with 25-μM fluorogenic peptide caspases 3 substrate rhodamine 110 bis-(N-CBZ-L-aspartyl-L-glutamyl-L-va-lyl-L-aspartic acid amide) (EnzChek^® Caspase-3 Assay Kit, Molecular Probes, Milano, Italy) at 25 °C for 30 min in a buffer solution composed by PIPES 10 mM pH 7.4, EDTA 10 mM, CHAPS 0.5%, modifying experimental protocol from [18 (link)]. The amount of cleaved substrate per sample was measured in a 96-well plate fluorescent spectrometer (PerkinElmer, Milan, Italy) setting the following wavelengths: excitation at 496 nm and emission at 520 nm.

To determine the levels of apoptosis, cells expressing the E proteins were analyzed using the colorimetric Enz-Chek Caspase-3 assay kit according to the manufacturer’s instructions (Molecular Probes, E-13183). HEK293 cells were either mock-transfected, transfected with pcDNA3.1( +), transfected with pcDNA3.1( +), and treated with 2 µM staurosporine, or transfected with the vectors expressing the E proteins. 48 h pt, cells were harvested, lysed, and centrifuged to clear cellular debris. The supernatants were collected and reacted with Z-DEVD-AMC substrate in a microplate for one hour at room temperature. Fluorescence was measured with the BioTek Synergy H1 microplate reader using excitation at 342 nm and emission at 441 nm. Samples were normalized to the total protein used in each sample. Caspase-3 activity in the pcDNA3.1( +) transfected sample was set as the baseline control.

To measure the caspase-like protease activity during erythrocytic stage in drug treated and untreated parasites EnzChek® Caspase-3 Assay Kit (Molecular probe) was used and assay was performed according to the manufacturer's instructions with some modification. In brief, infected erythrocytes (iRBCs) having 8–10% parasitaemia were exposed with 10 nM of ART/ARS and 100 nM of CDRI-97/78 for 24 h. Cell free parasites were prepared by saponin lysis of drug treated and untreated iRBCs. Parasite lysate were prepared by mild sonication and 2X reaction buffer containing caspase substrate Z-DEVD–R110 was added into 1:1 ratio followed by incubation at room (25°C) temperature for 30 min. in dark (Gunjan et al., 2016 (link)). Increase in fluorescence caused by cleavage of the Z-DEVD-R110 substrate was measured at excitation and emission wavelengths of 485/20 and 530/20 nm, respectively using micro plate reader (Synergy HT Biotek). In a parallel set of reactions, the caspase inhibitor Ac-DEVD-CHO was added to the reaction mixture before the addition of parasite lysate. All the experiments were performed in duplicate.

Caspase enzymatic activity assay was performed to determine the apoptotic effect of Met and Met-loaded liposomes. The assay was performed using an EnzChek^® caspase-3 assay kit (Molecular Probes, Eugene, OR, USA) as previously reported [34 (link),35 (link)]. This assay kit is based upon the fluorometric detection of a bright blue fluorescent product obtained by proteolytic cleavage of 7-amino-4-methylcoumarin-derived substrate Z-DEVD-AMC. Briefly, 1.0 × 10⁶ cells/plate were seeded in petri dishes followed by treatment for 6 h with Met, blank DO lipo, and Met DO lipo along with a control group maintained. After treatment, cells were washed twice with ice-cold PBS (pH 7.4) and were scraped to obtain a cell suspension. The obtained cell suspensions were centrifuged to get cell pellets, which were then lysed using lysis buffer. The lysed pellets were centrifuged to separate the cell debris and the supernatant obtained was transferred to 96-well plate. Equal volume of substrate was added to the supernatant and the fluorescence was measured at excitation/emission 342/441 nm. Control without enzyme was used to estimate the background florescence of substrate in each assay.