Ldh glotm cytotoxicity assay

The evaluation of pyroptosis inhibition was done using the LDH-Glo^TM Cytotoxicity Assay (Promega Co. Madison, WI, USA). Briefly, THP-1 cells were cultured for 2 days in 96-well plates to a density of 5 × 10⁴ 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. Then, 2 μL of 10% Triton X-100 was added to 100 μL of Vehicle-treated Cells Control for 5 min before collecting the samples for LDH detection. Supernatants were harvested and 20-fold diluted in the LDH storage buffer and kept at –70 °C until analysis. The LDH activity was measured using the LDH-Glo ^TM Cytotoxicity Assay (Promega Co., Madison, WI, USA) according to the manufacturer’s instructions. The % Cytotoxicity = 100 × (Experimental LDH Release − Medium Background)/(Triton X-100 LDH Release Control − Medium Background).

Bone marrow-derived macrophages were also seeded at density of approximately 100000 cells/cm² (2⋅10⁵ in a 24-well plate). Lactate dehydrogenase (LDH) activity in supernatants was measured using the LDH-Glo^TM Cytotoxicity assay (Promega #J2380) according to the manufacturer’s instructions. Luminescence was recorded with a GloMax^TM 96 microplate luminometer (Promega). Total amount of cells was inferred by lysing cells with Triton^TM X-100 at the end of the experiments and comparing the values to the ones of an included standard curve.

Lactic dehydrogenase release was measured in the GBM culture supernatants using the LDH-Glo^TM Cytotoxicity Assay (Promega, cat n. J2380), according to the manufacturer’s instructions. Briefly, 1.5 × 10⁵/well GBM cells were plated in six-well plates. At the following time points from radiation treatment (1 day, 7 days, 14 days, 21 days, and 28 days), cell supernatants diluted 1:25 in LDH storage buffer (200 mM Tris-HCl (pH 7.3), 10% Glycerol, 1% BSA in deionized water) were added to LDH detection reagent at 1:1 ratio and incubated at room temperature for 60 min. Luminescence was measured using the GloMax^® bioluminescent reader (Promega). Percent LDH release (%), was calculated with the formula: LDH release (%) = [(experimental LDH release value) − (background value)]/[(LDH release value in 10% Triton X-100-treated samples) − (background value)] × 100. Lactic dehydrogenase release was normalized on the total amount of protein (µg).