Glutamine colorimetric assay kit

A Glutamine Colorimetric Assay Kit (K556-100, BioVision) was used to measure serum glutamine levels. Blood was collected preoperatively, and none of the patients had received radiotherapy, chemotherapy or supplementation with glutamine before surgery. Serum was obtained by centrifugation and stored at − 80 °C until detection. A parallel sample well was set to eliminate the influence of the background signal, and each sample was tested twice. Glutamine levels were calculated from the standard curve.
Carcinoembryonic antigen (CEA) is a classic tumour biomarker for CRC, and the serum CEA test has been widely used to monitor the presence of primary CRC and recurrent CRC following radical resection in clinical practice. Radioimmunoassays were used to determine the CEA in serum at the Department of Nuclear Medicine in the China-Japan Union Hospital of Jilin University. The normal reference value was between 0.5 and 9.6 µg/L.

Cells were seeded at 5 × 10⁵ cells per well in 60-mm dish followed by 200 nM insulin treatment for 72 h. The collected cells were separated equally to measure intracellular glutamine levels and protein concentrations. Glutamine levels were measured by glutamine colorimetric assay kit (#K556, Biovision, Milpitas, CA, USA) according to the manufacturer’s instructions. Glutamine concentration is expressed as nmol/mg of protein.

Cells were seeded and cultured as described above. At indicated time points (including time 0), the conditioned medium was collected, cleared by centrifugation, and subjected to measurements of glutamine and ammonium concentrations using a glutamine colorimetric assay kit (BioVision, Milpitas, CA, USA) and an EnzyChrom™ ammonia/ammonium assay kit (BioAssay Systems, Hayward, CA, USA), respectively. All experiments were done in triplicates and the averages were chosen for fitting the model parameters.

The concentrations of L-glutamine in culture media were determined (Glutamine Colorimetric Assay Kit, Biovision, K556-100). Briefly, cells were seeded in 6-well plate and allowed to adhere overnight. Changed with the fresh medium and cultured for 12 hr and 24 hr, supernatant was achieved by centrifuge at 1000 rpm for 5 min. 40 μl sample supernatant with 60 μl reaction buffer was added to 96-well plate. After 1 hr incubation at 37°C, samples as well as standards absorbance were measured at OD450nm. Data were normalized based on the viable cell counts measured by cell counting.

To measure glucose uptake and extracellular lactate, 1–2 × 10⁵ cells were seeded into a six-well plate. Twelve hours later, the medium was replaced with 3 ml of complete medium and supernatants were collected 24 h later and analyzed for glucose and lactate content using the Glucose colorimetric assay kit II (Bio Vision) and the Lactate colorimetric assay kit II (Bio Vision), respectively, according to the manufacturer’s instructions. Glucose consumption was extrapolated by subtracting the measured glucose concentrations in the medium from the original glucose concentration (25 mM). Both glucose consumption and lactate production are normalized to the total cell number. To measure glutamine consumption, 1–2 × 10⁵ cells were seeded onto six-well plates. Twelve hours later, the medium was replaced with 3 ml of complete medium and supernatants were collected 24 h later and analyzed for lactate content and glutamine consumption using the Glutamine colorimetric assay kit (Bio Vision), according to the manufacturer’s instructions. Glutamine consumption was extrapolated by subtracting the measured glutamine concentrations in the medium from the original glutamine concentration (25 mM). The rates of glucose uptake, lactate production, and glutamine consumption were normalized to cell number.

Glutamine level was measured from mouse brain slices using a glutamine colorimetric assay kit (Biovision, CA). The brain slices were recovered for 30 min in a chamber with aCSF aerated with 95% O₂ / 5% CO₂ at 32 °C. Some slices were collected immediately (time 0) and some slices were transferred to a separate chamber with different conditions for 1-h incubation. The assay is based on the hydrolysis of glutamine to glutamate, which produces a stable signal for the colorimetric measurement of tissue glutamine. The signal is proportional to the concentration of glutamine in the brain slices. The background signal without hydrolysis enzyme mix was subtracted to exclude the contamination of glutamate in the tissue. As recommended by the manufacturer, the samples were deproteinized using 10 K cutoff spin column. Glutamine levels were normalized to protein concentration of the samples. The optical density was measured at 450 nm using a plate reader.