Pyruvate colorimetric fluorometric assay kit

Lactate concentration of cell extracts was measured using Lactate Colorimetric/Fluorometric Assay Kit (K607-100, Biovision, USA). Pyruvate concentration of cell extracts was measured using Pyruvate Colorimetric/Fluorometric Assay Kit (K609-100; Biovision, USA).

The concentrations of cellular pyruvate and NADPH were determined by the Pyruvate Colorimetric/Fluorometric Assay Kit and PicoProbe™ NADPH Quantitation Fluorometric Assay Kit, respectively (K609-100 and K349-100; BioVision, Milpitas, CA, USA). On 6 cm plates, HEK293T (1.5 × 10⁶ cells), MRC-5 (3 × 10⁵ cells), HFL-1 (3 × 10⁵ cells) and H1299 (3 × 10⁵ cells) and MCF-7 (6 × 10⁵ cells) were cultured for 24 h in medium containing 20 mM L-malate at 37 °C with 5% CO₂. Cells were treated with EA or MDSA for 48 h. After harvesting, cells were sonicated in 100 µL phosphate-buffered saline (PBS). After centrifugation, the supernatant was deproteinized using a 10 kDa spin column (Acrodisc® syringe filter, Pall Life Sciences) and analyzed using working mixtures in a 96-well plate with the Biotek® multi-mode microplate reader to detect fluorescence at Ex/Em=535/587 nm (Agilent, Santa Clara, CA, USA).

Overnight cultures of the ATCC 25922 wild-type and ΔyhjX mutant strains were diluted with fresh media (MHB with or without ½ MIC gentamicin, M9 minimal medium containing 0.4% glucuronate and M9 minimal medium containing 0.4% glucose) to a final OD600 of 0.2. The levels of pyruvate in fresh culture and in supernatants of E. coli cultures were determined before inoculation, 30 min after inoculation and 60 min after inoculation using a pyruvate colorimetric/fluorometric assay kit (BioVision). Each experiment was replicated three times. The experimental values were calculated from a standard curve.
Quantitative RT-PCR was also performed to compare the expression levels of yhjX in the wild-type E. coli strain in different media and to analyze the relationship between yhjX expression levels and extracellular pyruvate concentrations. The yhjX expression in E. coli growing in M9 containing glucose at the 30 min time point was set as the control.

HUVECs were cultured for up to 48 h in the presence or absence of COCO. Culture media was collected at 1, 3, 6, 12, 24, and 48 h, centrifuged at 16,260 g for 5 min, aliquoted, and stored at −80°C. Media was analyzed for glucose concentration using a BioProfiler 400 Analyzer (BioNova). For measurements of pyruvate, the culture media was analyzed using the Pyruvate Colorimetric/Fluorometric Assay Kit (BioVision) as per the manufacturer’s instructions.

Pyruvate levels were determined by the Pyruvate Colorimetric/Fluorometric Assay Kit (BioVision) in accordance with the manufacturer’s instructions. Fifty microlitre of sample was used for each determination. Next, 50 µL of reaction mix was added into the cell plate and incubated at room temperature for 30 min, protected from light. Fluorescence was measured using a Synergy H1 Reader (BioTek) at an excitation of 535 nm and an emission of 590 nm.

Warburg effect describes an energy shift from mitochondrial oxidative phosphorylation to aerobic glycolysis and is evaluated by measurements of glucose uptake, acetyl-coenzyme A (acetyl-CoA) synthesis, and lactate production. In cancer cells, pyruvate is converted to lactate in spite of the presence of oxygen. Glucose uptake, pyruvate and lactate production were determined using Glucose Uptake Colorimetric Assay kit (ab136955), Pyruvate Colorimetric/Fluorometric Assay Kit (K609–100), and Lactate Assay Kit II (K607–100), respectively, following the protocols provided by manufacturers (Biovision, K686–100, Milpitas, CA, USA). All determinations were done in triplicate.

Lactate and pyruvate levels in the medium was measured using Lactate Colorimetric Assay Kit (BioVision Incorporated, #K607) and Pyruvate Colorimetric/Fluorometric Assay Kit (BioVision Incorporated, #K609) according to the manufacturer’s instructions, respectively. In brief, FACS-enriched ENCCs (Passage 1−2) were seeded at 5 × 10⁵/well onto a fibronectin-coated 12-well plate and incubated overnight. On the next day, cells were treated with/without PKM2 inhibitor (1 μM), SAG (1 μM), or cyclopamine-KAAD (1 nM) for 1 h. The medium were then collected, and the levels of lactate or pyruvate were measured simultaneously. The levels were normalized to DNA concentration. The experiments were performed in triplicate.