Caspase glo 1 inflammasome assay

The evaluation of caspase-1 activity was done using the Caspase Glo 1 inflammasome assay (Promega Co. Madison, WI, USA). Briefly, THP-1 cells were grown for 2 days in 24-well plates to a density of 2 × 10⁵ cells/well in the presence of 10 nM PMA at 37 °C in a humidified 5% CO₂ incubator. PMA-supplemented medium was then discarded and macrophages were pretreated with fresh medium containing fractions at different concentrations for 1 h. Dexamethasone at a concentration of 20 µg/mL or 20 µM AC-YVAD-CHO treatments were used as controls. Cells were further incubated with LPS (Sigma, Saint-Louis, MO, USA) (at a final concentration of 1 µg/mL) for 3 h and then with 5 µM ATP (Invitrogen/Thermofisher, Waltham, MA, USA) for additional 1 h. The caspase-1 activity was measured using the Caspase-Glo 1 Inflammasome Assay (Promega Co., Madison, WI, USA) according to the manufacturer’s instructions. The 24-well plate containing treated cells was removed from the incubator and 50 µL of the supernatant in each well were transferred to the corresponding well of a new white 96-well plate. An aliquot of 50 µL of the Caspase-Glo 1 reagent was then added to each well and gently mixed on a plate shaker at 300 rpm for 30 s. The mixture was then incubated for 1 h at room temperature before measuring the luminescence on a Berthold Technologies luminometer.

Caspase-Glo® 1 inflammasome assays (Promega, USA) were performed to evaluate caspase-1 activity, following the instructions of the manufacturer. Briefly, cells were seeded in 96-well plates and Caspase-Glo 1 reagent was added to each well. Then, cells were incubated at room temperature for 1 h. Subsequently, cells were shaken for 5 min and incubated again at room temperature for 30 min. Victor X (PerkinElmer, USA) was used to detect luminescence.

Caspase-1 activity was determined using the Caspase-Glo® 1 Inflammasome Assays (Promega, catalog no. G9951) according to the manufacturer’s instructions. Briefly, day 19 microglia were plated at a density of 1.2 × 10⁵ cells/cm² on Matrigel-coated 96 well plates and incubated overnight. The next day, cells were treated with 100 ng/ml of LPS (Sigma Aldrich, catalog no. L6529) for 3 h and subsequently with 5 mM ATP (Sigma Aldrich, catalog no. A2383) for 30 min at 37 °C and 5% CO₂. The culture supernatant was transferred into a white 96 well plate and Caspase-Glo® 1 Reagent was added. The mixture was incubated at room temperature in the dark for 1 h and luminescence was measured on a Tecan Spark microplate reader.