Flica caspase 3 7 assay kit

Caspase-3/-7 activity was determined using a FLICA Caspase-3/-7 assay kit (ImmunoChemistry Technologies, LLC, CA, USA). SAS cells were seeded at 5 × 10⁵ cells/well in a 24-well plate and incubated with or without docetaxel (100 nM) for 48 h in the absence or presence of SP600125 (20 μM), PD98059 (20 μM), or compound c (10 μM) at 37 °C (1 h pretreatment). At the end of treatment, cells were washed twice with PBS and stained with fluorescent probes for 30 min in a dark environment at 37 °C. After incubation with the dye, cells were carefully removed supernatants and washed twice with 1× wash buffer. The fluorescence intensity was measured using a spectrofluorometer (Gemini XPS Microplate Reader, Molecular Devices, San Jose, CA, USA) at an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Caspase-3/7 was estimated with the FLICA Caspase-3/7 Assay Kit (ImmunoChemistry Technologies, Bloomington, MN, USA). Both DLD-1 and HT-29 cell lines were incubated for 24 h with the tested compounds and reference drugs at 1.5 and 3 µM. The test was performed as described in the literature [23 (link)]. The data were collected and evaluated with FACSDiva software (version 6.1.3, BD Biosciences Systems, San Jose, CA, USA).

In vivo caspase-3/7 activity was measured by FAM-fluorochrome inhibitor of caspases (FLICA) Caspase 3&7 Assay Kit (Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer's information. Briefly, cells were incubated with the FAM-DEVD-fluoromethylketone (FMK) reagent for 1 h at 37 °C. After washing with PBS, cells were fixed with 0.1% (w/v) formaldehyde solution. In vivo CTSB activity was performed using Magic Red CTSB Activity Kit (Immunochemistry Technologies) according to the manufacturer's protocol. Cells were incubated with Magic Red CTSB Substrate (Immunochemistry Technologies) for 1 h at 37 °C. The cells were pelleted after centrifugation, washed with PBS, and fixed with 0.1% formaldehyde. Fluorescence was measured by a fluorescent microplate reader Infinite 500 (Tecan Group Ltd., Männedorf, Switzerland), or at least 200 cells were counted and indicated as the percentage of positive cells per total cells. Images were obtained by fluorescent microscopy Leica DMRB or confocal microscopy Leica TSC SP2 (Leica Microsystems, Wetzlar, Germany).