Lactate glo assay

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.

Glucose, lactate, glutamine, and glutamate concentration were measured using Glucose-Glo™ Assay, Lactate-Glo™ Assay and Glutamine/Glutamate-Glo™ Assay (Promega), respectively. Culture medium dilution of 1/500, 1/100 and 1/50 in PBS were used to measure glucose, lactate, and glutamine/glutamate concentration, respectively. Cell ATP concentration was measured using CellTiter-Glo® Luminescent Cell Viability Assay (Promega), following the manufacturer’s instructions. Cell DNA content was measured using CyQUANT™ Cell Proliferation Assay (Invitrogen) by resuspending cell pellets in 250 μL CyQUANTTM GR dye/cell-lysis buffer. Finally, cell protein content was measured by resuspending cell pellets in PathScan® Sandwich ELISA Lysis Buffer (Cell Signalling Technology) then by using Pierce™ 660 nm Protein Assay Reagent (Thermo Scientific™) following the manufacturer’s instructions. For all these assay, absorbance, fluorescence, and luminescence were read with SpectraMax® M3 plate reader (Molecular Devices).

The Lactate-Glo Assay (J5021; Promega) was used to measure lactate levels^{52 (link)}. After homogenizing ten larval CNSs in 100 µL of PBS, samples were centrifuged at 12,000×g at 4 °C for 10 min, and the supernatant was transferred to a new tube. In each well of a microtiter plate, 1 µL of sample, 49 µL of PBS, and 50 µL of lactate detection reagent were mixed and the luminescence was read on a Lumat LB 9507 luminometer.

The levels of lactate in zebrafish hepatic tissues were determined using the Lactate-Glo™ Assay (Promega, J5021). Luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega). We used the Pyruvate Assay Kit (BestBio, BB47421) to detect pyruvate in hepatic tissues and the results were obtained using the MD-SpectraMax M5. All procedures were performed according to the manufacturer’s instructions.

From isolated CD4⁺ T cells and monocytes, 30 000 cells were used for metabolite detection in duplicates for each sample. Lactate-Glo assay (Promega, #J5022; Promega, Madison, Wisconsin, USA), Glutamate-Glo assay (Promega, #J7022), and Glucose-Glo assay (Promega, #J6022) were used to measure intracellular metabolites according to the manufacturer's instructions. Luminescence was measured using Varioskan microplate reader (ThermoFisher Scientific, Waltham, Massachusetts, USA).

After the treatment, the cell culture medium was collected. LDH activity was performed using the LDH activity assay kit (Sigma-Aldrich) following the manufacturer’s instructions. The absorbance was measured at 450nm with the Tecan Infinite F Plex at 37°C every five minutes, for 26 cycles. LDH activity of the treatments samples were compared to the untreated controls.
Lactate concentration was measured in the cell culture medium using the LactateGlo assay (Promega) according to manufacturer’s instructions. Luminescence was measured with the Tecan Infinite F Plex. Lactate concentrations of the treated samples were compared to the untreated controls.
The LDH activity and lactate concentrations were normalized based on cells amount, OMS size, and OMS size with invasion area, in the 2D, 3D-SUSP and 3D-OOAC cell cultures, respectively.