Caspase 3 7 fluorescence assay kit

The level of PCD in M. aeruginosa exposed to D. magna and D. cucullata extracts was evaluated using a Caspase-3/7 Fluorescence Assay Kit (Cayman Chemical, United States) (Hu and Rzymski, 2019 (link)). This assay employs a specific substrate, N-Ac-DED-N′-MC-R110, which upon the specific proteolytic cleavage of the amino acid sequence Asp-Glu-Val-Asp by active caspase-7 and/or caspase-7 generates a fluorescent rhodamine-110. The 5 ml subsample of incubated cells were washed twice with the provided Assay Buffer (800 × g, 5 min), incubated with a cell-lysis buffer based on 1% Triton X-100 (Cayman Chemical, United States) for 30 min on an orbital shaker at 21°C, washed again and mixed with caspase-3/7 substrate solution. The fluorescence signal was recorded with a Synergy HTX Multi-Mode Microplate Reader (BioTek, USA) at an excitation of 495 nm and an emission of 528 nm. The results were calculated as the percentage of fluorescence intensity of the control sample.

Caspase 3, caspase 7 and caspase 9 activation was measured using a Caspase 3/7 Fluorescence Assay kit (Cayman Chemical, Ann Arbor, MI, USA) and a Caspase 9 fluorometric assay kit (Enzo Life Science, Roma, Italy). The results are expressed as nmol of the hydrolyzed substrate of each caspase/mg cellular proteins, according to a previously set titration curve.

Caspase 3/7 activity was analyzed using a Caspase-3/7 fluorescence assay kit (Cayman Chemical; Ann Arbor, MI, USA) following the manufacturer’s protocols. In brief, 10⁴ NHEK-Ad cells were plated into 96-well cell culture plates (SPL). At 80% confluence, the cells were irradiated with 120 mJ/cm² UV light with or without pre-treatment of DeinoPol (10 μg). After 3 h, cells were lysed, followed by incubation of 90 μL cell lysate and 10 μL caspase 3/7 substrate solution. The fluorescence intensity of each well was measured with excitation 485 nm and emission 535 nm using a Victor X3 light plate reader (PerkinElmer).

Cells were seeded, treated with Granulocystopsis sp. methanol extract at their respective IC₅₀ values, and incubated for 48 h. Caspases activity was then determined using Caspase-3/7 Fluorescence Assay Kit (Cayman cat. No. 10009135) (Martinotti, Ranzato & Burlando, 2018 (link)) according to the manufacturer’s instructions. Three independent experiments were performed with three technical replicates each.

Cells were plated in 96-well plates (8000–12,000 cells/well) and allowed to adhere overnight. Cells were treated with LLL12B or DMSO only. After 5 h treatment, active caspase-3/7 were measured using Caspase-3/7 Fluorescence Assay Kit (Cayman, Ann Arbor, MI) according to the manufacture’s instruction. Briefly, cells were centrifuged in the plate (800 g, 5 min) and the culture medium was aspirated, and then 200 µL of assay buffer was added to each well to wash the cells and the supernatant was removed after centrifuge. After that, 100 µL of cell-based assay lysis buffer was added to each well with shaking for 30 min at room temperature. Subsequently, 90 µL of the supernatant from each well was transferred to a corresponding well in a new black 96-well plate and 10 µL of assay buffer was added to appropriate wells. The plate was incubated at 37 °C for 90 min followed by addition of 100 µL of the caspase-3/7 substrate solution. Finally, the fluorescence intensity of each well was read at excitation 485 nm and emission 535 nm. All treatments were performed in triplicate.

The Caspase-3/7 Fluorescence Assay Kit (Cayman, Ann Arbor, MI, USA) was used to detect the activation of caspase-3/7 and the assay was performed according to the manufacturer’s instructions. Briefly, MDA-MB-231, SUM159, and 4T1 cells were seeded in a 96-well plate in triplicate. After overnight incubation, the cells were treated with either LLL12B or DMSO for 5 h and then lysed and processed to measure caspase-3/7 activity and the fluorescence intensity was read (excitation = 485 nm; emission = 535 nm).