Caspase 3 multiplex activity assay

Caspase activity was quantified using the fluorometric kit Caspase 3 Multiplex Activity Assay (ab219915, Abcam) according to the manufacturer’s protocol. Cells from the PDL cell line PDL26 were grown in a 96-well plate at 2 × 10⁴ cells per well. The cell line was obtained from the third molar of a healthy 26-year-old male non-smoker after written consent according to the ethics regulations of the University of Goettingen (file no.: 27/2/09). Immortalization with human Telomerase Reverse Transcriptase has been described before [64 (link)]. The cells were treated with 10 nM staurosporine (Sigma-Aldrich, Munich, Germany), 60 ng/mL SOD2 (Abcam), 60 ng/mL BIRC3 (Abcam), or staurosporine in combination with SOD2 or BIRC3 for 1 d. Untreated cells served as control. Then, 100 μL/well of caspase assay loading solution was added to each well, and the plate was incubated at room temperature for 60 min and protected from light. Subsequently, the fluorescence intensity was measured using a SpectraMax iD5 Multi-Mode microplate reader (Molecular Devices, CA, USA) at specific wavelengths (Ex/Em = 535/620 nm). For data analysis, the blank readings were subtracted from all measurements, and the CASP3 activity of each group was determined in relation to the control group.

The Caspase-3, -8 and -9 activities were assessed using the fluorometric kit Caspase 3 Multiplex Activity Assay (Abcam), according to the manufacturer’s instructions.
Briefly, the RD or RH30 cells (2 × 10⁴ cells/well) were plated in 96-well black plate and after 24 h treated with 500–1000 µM FAC or 100–200 µM DFP for 3–6–24–48–72 h. To assay the Caspases activity in each well, an Assay Loading Solution was prepared by adding 50 µL of substrate (Caspase-3, -8, -9) to 10 mL of Assay buffer. Then, 100 µL of the Caspases Assay Solution were added to each well, without removing culture media. The plate was incubated at room temperature for 60 min, protected from light and the fluorescence (RFU) was monitored by the EnSight Multimode plate reader (PerkinElmer, Waltham, MA), at specific wavelengths, as follow: Ex/Em = 535/620 nm (Caspase-3), Ex/Em = 490/525 nm (Caspase-8), Ex/Em = 370/450 nm (Caspase-9).