Malate assay kit

Secreted metabolites were measured using the Glucose-Glo Assay, the Lactate-Glo Assay, the Glutamine/Glutamate-Glo Assay (all Promega) as well as the Fumarate Detection Assay and the Malate Assay Kit (both Abcam) following the manufacturer’s instructions. To avoid additional background signals, the hydrogels were cultured in phenol-red free medium supplemented with dialyzed FBS (A3382001, Thermo Fisher) for 19 days. Hydrogels were washed three times with PBS and fresh medium was added. Supernatant of hydrogels was collected and snap-frozen 6 h, 24 h, 48 h and 72 h after the medium change. For glucose, lactate and glutamine/glutamate detection samples were diluted at least 1:50 for measurement. Samples or sample dilutions, media controls and standard dilutions were prepared and mixed with assay buffer as described by the manufacturers. The resulting luminescence (Glucose-Glo Assay, Lactate-Glo Assay, Glutamine/Glutamate-Glo Assay) or absorbance (Fumarate Detection Assay, Malate Assay Kit) were measured on a FLUOstar Omega plate reader (BMG Labtech Ltd). For quantification, standard titration curves were prepared for glucose, lactate, glutamine, glutamate, fumarate and malate and included on each plate. Medium samples were measured and subtracted from all samples as background.

Mice were fasted by removing diet at the beginning of the light cycle (06:00), and were CO₂-euthanized after 6 h (12:00). Liver was rapidly removed and diced into small pieces, rinsed in PBS, and flash frozen. Enriched liver subcellular fractions were deproteinized (BioVision; #K808) according to kit instructions. Specific assays were used to determine the concentrations of acetyl-CoA (Abcam Acetyl-CoA Assay Kit #ab87546), citrate (Abcam Citrate Assay Kit #ab83396) and malate (Abcam Malate Assay Kit #ab8391). Assays were run according to kit instructions.