Synergy htx 96

Cells were washed with fresh medium, resuspended at 1 × 10⁵ cells/ml in 0.2 ml of 100 mM TRIS 10 mM/EDTA I mM (pH 7.4), and sonicated on ice in two 10 s bursts. Tumour homogenates were resuspended in the same buffer and sonicated. Enzymatic activity in cell lysate was measured after incubation for 5 min at 37 °C. The protein content was measured using a BCA1 kit (Sigma, St. Louis, MO). The activity of PFK was measured spectrophotometrically following the procedure reported in [100 (link)]. ALDO activity was measured by using an Aldolase Activity Colorimetric Assay Kit (BioVision, Milpitas, CA). The activity of GAPDH, ENO and LDH was measured spectrophotometrically according to the procedure described in [101 (link), 102 (link)]. For the GAPDH activity measurement, the cell lysate was incubated with 5 mM 3-phosphoglyceric acid, 1 U phosphoglycerate 3-kinase, 5 mM ATP and 2.5 mM NADH. For the ENO activity measurement, the cell lysate was incubated with 10 mM MgCl₂, 100 mM KCl, 1 mM 2-phosphoglyceric acid, 0.4 mM ADP, 6.8 U/mL Pk, 9.9 U/mL Ldh, and 0.2 mM NADH. The PK activity was measured with a pyruvate kinase assay kit (Abcam, Cambridge, UK). For all assays of glycolytic enzymes, the activity levels were monitored by measuring the absorbance variation at 340 nm with a Synergy HTX 96-well microplate reader (Bio-Tek Instruments).

Cellular ATP levels were measured using a luminescent ATP detection assay kit according to the manufacturer’s instruction (ab113849, Abcam, Cambridge, MA). Cells were seeded at a density of 3,000 cells/well, allowed to adhere for 4 h, and dosed in the same method as the cytotoxicity assays. Luminescence of luciferase was read via a BioTek Synergy HTX 96-well microplate reader (Biotek, Winooski, VT). ATP concentration for individual wells was interpolated via standard curve in GraphPad Prism (GraphPad Software, Santa Clara, CA). Luminescence of cell culture media was subtracted from each well. Data was then averaged, corrected for proliferation, and subsequently normalized to their respective controls. Biological replicates were performed in technical triplicate. Data shown are from n = 2 independent biological replicates.

The opening of the mPTP, considered an index of damaged mitochondria, was measured using a Mitochondrial Permeability Transition Pore Assay Kit (BioVision) according to the manufacturer’s instructions. The intracellular fluorescence was measured at an excitation λ wavelength of 488 nm on a Synergy HTX 96-well microplate reader (Bio-Tek Instruments). The results are expressed as relative fluorescence units (RFU)/mg cellular proteins.