Caspase 3 activity kit

Caspase-3 activity was measured using a Caspase-3 activity kit in accordance with the manufacturer's instructions (Biovision). Cells were collected and washed 3 times in ice-cold PBS, re-suspended in lysis buffer, incubated on ice for 20 min, and then centrifuged at 12,000g at 4°C for 10 min. The supernatant was kept and protein concentration was quantified using a BCA protein quantification kit (Thermo). Then 100 μg of protein extracts were diluted with dilution buffer and incubated with DEVD-pNA substrate at 37°C for 1 h. The samples’ absorbance was measured at 400 nm using a TECAN Infinite M1000 microplate reader.

The cells were routinely treated as mentioned above. The activity of caspase-3 in cells was measured using a caspase-3 activity kit according to the manufacturer's protocol (BioVision Inc., Milpitas, CA, USA). In brief, cytosolic proteins (200 μg in 50 μL) were mixed with the caspase-3-specific substrate Ac-DEVD-pNA (Jiancheng, Nanjing, China) and incubated at 37°C for 4 h. The absorbance was measured at 405 nm with an enzyme marker.

Because caspase induction has been considered a representative programmed cell death response due to oxidative stresses (Bhattacharjee and Mishra, 2020 (link)), the caspase activity of M. aeruginosa NIES-298 cells was assessed using a caspase-3 activity kit (Biovision, United States), according to the manufacturer’s instructions. Briefly, M. aeruginosa cells were treated with 25 and 50 μM mannitol and 1 mM H₂O₂ (as a positive control) and were incubated under the same culture conditions described above. Cells were lysed using a lysis buffer and incubated with the caspase-3 substrate, DEVD-AFC (7-amino-4-trifluoromethyl coumarin), at 37°C for 1 h. Fluorescence was measured at 404 nm excitation and 505 nm emission and was normalized to the OD values at 680 nm. To assess oxidative stress generation by mannitol treatment, 30 ml of NIES-298 cell cultures in 60-ml air-filtered culture bottles were treated with superoxide dismutase (SOD, 50 U⋅ml^–1), catalase (50 U⋅ml^–1), and glutathione (1 mM), along with mannitol (50 μM), and their growth was assessed by measuring OD (680 nm).