Glutamine glutamate glo assay

CellTiter-Glo® 2.0 Assay Kit was used to determine the ATP amount in cell lysates. Glucose-Glo™ Assay Kit and Lactate-Glo™ Assay Kit (Promega, Madison, WI) were used to measure glucose uptake and lactate levels in cell supernatants, respectively. Glutamine/Glutamate-Glo Assay (Promega) was used to assess the changes in glutamine and glutamate content in cells with indicated treatments. Changes in the level of total intracellular GSH were determined using Glutathione Detection Assay Kit (Fluorometric, Abcam, MA). For GSH determination, HN6 and HN31 cells were treated with or without 100 μM CPI-613 for 24 h, and the lysates were collected and incubated on ice for 30 min. The supernatants were collected after centrifugation at 12,000×g for 10 min and processed according to the kit protocol, and fluorescence was measured at 380/461 nm (excitation/emission wavelengths).

For the luminescent assays, the MEC-1 and HG-3 cells were seeded in triplicate with CB-839 (0.1-100 μM). After 72 hours of incubation, cells were counted and 50,000 cells per each condition were taken for the luminescent assays. To determine the intracellular glutamate concentrations, a Glutamine/Glutamate-Glo Assay (Promega) was used following the manufacturer’s instructions. The reduced glutathione levels were measured using a GSH-Glo Assay (Promega) according to the manufacturer’s instructions. Luminescence was measured using a BioTek Synergy HT microplate reader. Metabolite concentrations were calculated using a standard curve method.

DC were treated as above, and after 17 hours, cells and supernatants were collected alongside blank medium as controls. Extracellular glucose was quantified with a glucometer against glucose standards essential as previously described [1 (link)]. Glucose levels (mg/dl) in the blank medium were determined alongside DC +/- treatments. Glucose uptake was calculated by subtracting the glucose in the control, IAV, IAV^BPL, PolyIC, R848, or LPS medium from the cell-free blank medium. Internal and external glutamine were quantified from supernatant and cell lysates, respectively, following the Glutamine/Glutamate Glo assay manufacturer's protocol (Promega, Madison, WI). Briefly, samples were prepared in PBS, diluted to fit in to the linear range of the glutamine or glutamate standard curve, and.25 μl sample or standard transferred to a 96 well luminescence assay plate and reaction allowed to proceed for 30 minutes at room temperature. Next, substrate detection reagent was added and incubated for an additional 60 minutes and metabolite detection was performed using a bioluminescent NADH detection method with luminescence measured using a BMG CLARIOstar microplate reader.

For measuring changes in glutamine and glutamate in the CSC medium, CSCs were isolated from MDA231, MCF-7, PC-3, and LnCap cells and plated in SFM conditions in low adherent 96-well plates at a density of 10,000 cells/cm². CSCs were grown in 100 μL DMEM medium supplemented with variable glucose concentrations, 4 mM glutamine, and growth factors (see Section 4.2, CSC isolation). The cells were incubated in a tissue culture incubator (37 °C, 5% CO₂), and were treated with sFRP4 (250 pg) on the 3rd day for 24 h. At the indicated time (i.e., 24 h treatment), 2 μL of culture medium was removed and transferred to a separate 96 well-plate containing 98 μL PBS/well. For metabolite analysis, 4.5 μL of thawed sample was transferred to respective 96 well white luminescent plates for glutamine and glutamate detection. Samples were then assayed as described in Glutamine/Glutamate-Glo Assay (#J8021, Promega) standard protocols [77 (link)]. Luminescence was read on a luminometer (EnSpire Multilabel Plate Reader, Perkin-Elmer).