Glomax bioluminescent reader

Cell viability of Panc-1 spheroids was measured using a 3D cell viability assay (Promega, Milan, Italy). Briefly, homogeneous spheroids were removed from the 96-well low-attachment culture plate and placed separately in single wells of a 96-well opaque culture plate (BD Falcon). CellTiter-Glo^® 3D reagent was added to each well and the luminescence signal was read after 30 min with the GloMax^® bioluminescent reader (Promega).

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).

Two thousand HCC cells/well or 1500 CCA cells/well were seeded into polyHEMA-coated 96-well culture plates. Upon formation, spheroids were stimulated immediately (time 0) with recombinant human ANG-2 (rh-ANG-2) (623-AN, R&D Systems, Minneapolis, MN, USA) and/or recombinant human VEGF (rh-VEGF) (293-VE, R&D Systems) proteins at increasing doses (0, 50, 100, 200, 400, 800 ng/mL). After 48 h from stimulation, the 3D viability assay was performed by transferring the obtained spheroids to a blank 96-well plate without coating and then adding the endpoint CellTiter-Glo^® 3D reagent (Promega, Milan, Italy) to induce the ATP release from spheroids, according to the manufacturer’s instructions. The luminescence signal was recorded after 30 minutes with the GloMax^® bioluminescent reader (Promega).