Flica apoptosis detection kit

Unless otherwise stated, all chemicals were purchased from Sigma (St Louis, MO, USA). Stock solutions of amitriptyline (100 mM), bupropion (100 mM), citalopram (10 mM), EGTA (50 mM), fluoxetine (10 mM), and N-acety-l-cysteine (NAC, 300 mM) were prepared in distilled water (DW). 5-fluorouracil (100 μg/mL), doxorubicin (2 mg/mL), paroxetine (20 mM), PD98059 (20 mM), SB203580 (20 mM), SP600125 (20 mM), taxol (1 mg/mL), tianeptine (100 mM), and Z-VAD-FMK (10 mg/mL, InvivoGen, San Diego, CA, USA) were dissolved in dimethyl sulfoxide (DMSO). The solutions were then diluted in culture medium to the working concentration. Where DMSO was used as a solvent, solution containing equivalent concentration of DMSO was used as a control. Final DMSO concentrations for chemicals indicated above were ~0.1% (v/v).
Dulbecco’s modified Eagle’s medium (DMEM/F12), DMEM, fetal bovine serum (FBS), and horse serum were purchased from Gibco-BRL (Gaithersburg, MD, USA). RPMI-1640 was purchased from Lonza (Walkersville, MD, USA). Total glutathione assay kit was purchased from Assay Designs (Ann Arbor, MI, USA). FLICA apoptosis detection kit was purchased from Immunochemistry Technologies (Bloomington, MN, USA). MitoSox was purchased from Invitrogen (Carlsbad, CA, USA). JC-1 mitochondrial membrane potential detection kit was purchased from Biotium Inc (Hayward, CA, USA).

Caspase activity was determined with the FLICA Apoptosis Detection Kit (Immunochemistry Technologies, Bloomington, MN, USA) according to the manufacturer’s instructions. Cells were treated with the examined compounds for the indicated periods of time after which cells were collected and suspended in a buffer containing the caspase inhibitor—a carboxyfluorescein-labeled fluoromethyl ketone peptide. Cells were subsequently incubated for 1 h 37 °C under 5% CO₂ cells and next were washed with washing buffer. The fluorescence intensity of fluorescein was determined with flow cytometry (BD FACSCalibur) and caspase activity was determined as the amount of fluorescence emitted from caspase inhibitors bound to the caspases.

To examine the induction of caspase activity by ramentaceone, the FLICA Apoptosis Detection Kit (Immunochemistry Technologies) was used. The kit uses FLICA (Fluorochrome Inhibitor of Caspases), a carboxyfluorescein-labeled fluoromethyl ketone peptide, which irreversibly binds to many active caspases. Caspase labeling was performed according to the manufacturer’s instructions. Briefly, cells were treated with ramentaceone (0–15 μM) for 12 h after which they were collected and suspended in a buffer containing the caspase inhibitor. After a 1 h incubation at 37°C under 5% CO₂ cells were washed with washing buffer and the fluorescence intensity of fluorescein was determined with flow cytometry (BD FACSCalibur). Caspase activity was determined as the amount of fluorescence emitted from FLICA probes bound to the caspases.

The activity of caspases 3/7, caspase 8, and caspase 9 in PC3 cells treated with B2 was detected using the carboxyfluorescein FLICA apoptosis detection kit [15 ] following the manufacturer's instructions (Immunochemistry Technologies, LLC). The percent of caspase induction (CI) was calculated.