Colorimetric l lactate assay kit

HmoDCs were stimulated with control excipients, V132 (3 FTU per ml), MV140 (10⁷ bact. per ml) or MV140/V132 (MV140, 10⁷ bact. per ml, and V132, 3 FTU per ml) for 18 h. Glucose concentration in cell-free supernatants was determined by using the Glucose (GO) Assay Kit (Sigma-Aldrich). The metabolic rate was derived mathematically in percentage of medium without hmoDCs (2 mg/ml). Lactate production in cell-free supernantants was determined by using the colorimetric L-Lactate Assay kit (Sigma-Aldrich). The mitochondrial membrane potential was measured using MitoTracker Red Mitochondrion-Selective Probe kit (ThermoFisher Scientific) following the manufacturer’s instructions with minor modifications. Briefly, mitochondrial activation was measured using MitoTracker Red CMXROS (250 nM) for 30 min at 37°C after 18 h of treatment with the corresponding stimulus. The fluorescence of the probe was analyzed using FLUOstar OPTIMA fluorescence reader (BMG LabTech).

Standard enzyme reagent kits were used to measure relative glucose and lactate concentrations. Spheroids generated in different conditions were obtained 3 and 7 days after seeding the HCT116 cells. Spheroid cultures were transferred and incubated in PBS for 2 h. Then the culture medium (glucose content of 4.5 g/L) was changed and incubated for an additional 12 h to measure relative glucose/lactate levels; 200 μl medium were taken from each sample and analyzed using the Glucose (GO) Assay Kit (Sigma/GAGO20-1 KT) and the colorimetric l-lactate assay kit (Sigma/MAK329-1 KT). The absorbance at 540 nm for the glucose assay and at 570 nm for the lactate assay was measured in a Synergy H1 multimode microplate reader (Biotek). Spheroid samples were lysed in 1% (m/v) sodium dodecyl sulfate (SDS, Sigma) solution in PBS, and protein concentration was determined with standard bicinchoninic acid (BCA) assay (Thermo Scientific). Glucose consumption and lactate production rates in spheroid cultures were determined and compared with total protein concentration.