Muse multicaspase assay kit

The detection of multiple caspase (caspase-1, 3, 4, 5, 6, 7, 8 and 9) activation was done using Muse™ multicaspase assay kit (Merck Millipore, USA), following the manufacturer’s instruction. Briefly, 1 × 10⁵ cells/well/1 mL culture media were seeded at 37 °C in a 12-well plate for 24 h. The cells were then treated with MP-HX and MP-EA extracts for 24 or 48 h. The concentration of the extracts used were at ~IC₅₀ values for HCT116, HCC1937, HepG2 and MDA-MB-231 cells, where MP-HX concentrations were 70 μg/mL, 90 μg/mL, 75 μg/mL, and 45 μg/mL, respectively, while MP-EA concentrations were 75 μg/mL, 90 μg/mL, 130 μg/mL and 90 μg/mL, respectively. After treatment with the extracts, the cells were resuspended in 1X caspase buffer and 50 μL of the cells were transferred to 1.5 mL microcentrifuge tubes. Five μL of Muse™ multicaspase reagent working solution was added to the cells and incubated for 30 min in a 37 °C incubator. Then, 150 μL of Muse™ caspase 7-aminoactinomycin D (7-AAD) working solution was added in each tube and mixed and incubated in the dark for 5 min at room temperature. The percentage of cells with multicaspase activity was then measured using Muse™ cell analyzer (Merck Millipore, USA) flow cytometer.

Multicaspase activity percentage was quantified using a Muse MultiCaspase Assay Kit (Merck Millipore, USA). MDA-MB-231 cells (5 × 10⁵ cells/well) were seeded into 6-well plate for 24 h in triplicate and treated with 30 and 60 μg/ml of F1 fraction for 48 h. The Muse Cell Analyzer (Millipore, USA) was used to evaluate multi-caspase activity according to manufacturer’s directions.

Multicaspases activity was quantified by using the Muse MultiCaspase assay kit (Merck Millipore). MC3T3‐E1 cells were pre‐treated with 0.5% and 1% of methanolic extract from Mn(II)‐enriched C. glomerata for 24 hours and exposed to 1 µg/mL of LPS, and multicaspase activity was subsequently assessed according to the manufacturer's instructions, using the Muse Cell Analyzer (Millipore).

A stock solution of Fraisinib in DMSO 10 mM was diluted 1:100 with a culture medium. 20 μL of this solution was added to the wells containing the cells in 180 µL of culture medium to obtain a final concentration of 10 µM for Fraisinib and 0.1% DMSO. A mixture of the culture medium containing the same amount of DMSO but without Fraisinib was used for the sham test indicated as “DMSO.” Therefore, the concentration of DMSO in the cell wells was identical for Fraisinib-treated and DMSO-treated cells. An additional set of cells were not treated with any solution, and they are referred to in the main text as control (see Figure 2A).
After 48 h treatment, the Muse™ MultiCaspase Assay Kit (Millipore Merck, Vimodrone MI, Italy) was used for the detection of multiple caspase activation (caspase-1, -3, -4, -5, -6, -7, -8, and -9), following the manufacturer’s instructions. The percentage of cells with multicaspase activity was then examined using a Muse™ Cell Analyzer (Millipore Merck, Vimodrone MI, Italy) flow cytometer. The signal intensity for each antigen-specific antibody spot is proportional to the relative concentration of the antigen, and thus of the protein, in the sample.

Caco-2 cells (1 × 10⁶ cells/well/ 2 mL culture media) were seeded at 37 °C in a 6-well plate, when cells reached 70% of confluence, we treated them with Dox (IC₅₀) or ME (IC₅₀) or ME (IC₁₀) combined Dox (IC₅₀) for 24 h. A Muse™ multicaspase assay kit (Millipore Merck, Vimodrone MI, Italy) was used for the detection of multiple caspase activation (Caspase-1, 3, 4, 5, 6, 7, 8 and 9), following the manufacturer’s instruction. Then, the percentage of cells with multicaspase activity was examined using a Muse™ cell analyzer (Millipore Merck, Vimodrone MI, Italy).

The percentage of cells with multi-caspase activity was determined using a Muse^®® multi-caspase assay kit (Catalog No. MCH100109, Merck Millipore), following the manufacturer’s instructions. In this assay, the experiment was carried out in the same way as the apoptosis assay. After treatment, the treated cells were harvested in 1X caspase buffer. The Muse™ multicaspase reagent and Muse™ caspase 7-aminoactinomycin D (7-AAD) working solution were stained in each sample. The multi-caspase activity was performed following the protocol provided by the manufacturer, and analyzed using the MUSE^®® Cell Analyzer [19 (link)].

Muse^® MultiCaspase Assay Kit (Merck, Germany) was engaged in order to quantify the number of cells with caspase activity. This assay was based on the employment of Fluorescent-Labeled Inhibitor of Caspases (FLICA) allowing for the detection of the presence of activity of multiple caspases (caspase-1, 3, 4, 5, 6, 7, 8, and 9). In this set of experiments breast, cancer MCF-7 cells were treated with tested agents for 24 hours at the dose of 20 µg/mL. For the purpose of the clarity of presented data, live cells with no detected caspase activity (FLICA-negative and 7-AAD-negative) were not presented in the provided figures.