Glucose assay kit wst

Glucose and lactate concentrations in medium were determined by Glucose Assay Kit-WST (Dojindo^®, G264) and Lactate Assay Kit-WST (Dojindo^®, L256), respectively. LASCPC-01 cells were plated in 6-well plates in modified HITES medium. After incubation for 24 hours, cultured media were centrifuged at 1,500 rpm for 5 minutes, and supernatants were collected in 96-well plates in 100μl/well in triplicates. The supernatants were then incubated with corresponding respective reaction buffers at 37°C for 30 minutes, and then the absorbance was measured by a plate reader at 450 nm. The mass of glucose consumption and lactate production was normalized to the average number of cells at the start and end of incubation.

The intracellular contents of glutathione, glucose, and lactate were measured using GSSG/GSH Quantification Kit, Glucose Assay Kit-WST, and Lactate Assay Kit-WST (Dojindo Laboratories, Kumamoto, Japan), respectively, according to the manufacturer’s instructions.

HepG2 cells (5 × 10⁴ cells/well) were cultured in 48-well plates. Extracellular glucose and lactate were quantified using a Glucose Assay Kit-WST (Dojindo Laboratories, #G264) and a Lactate Assay Kit-WST (Dojindo Laboratories, #L256), respectively. Experiments were performed according to the manufacturer’s instructions. Briefly, 4 out of 200 μl of cultured medium were diluted in 196 μl of ultrapure water. Fifty or twenty microliters of diluted medium was used for the Glucose Assay Kit-WST or Lactate Assay Kit-WST, respectively. Samples were collected serially from the same wells at 12, 24, and 48 h after medium change. Cell survival was quantified by using Cell Counting Kit-8 (Dojindo Laboratories, #CK04) according to the manufacturer’s instructions.

Intracellular glucose content was determined using the Glucose Assay Kit-WST (G264, Dojindo Molecular Technologies, Inc., Rockville, MD, USA), according to the manufacturer’s protocol. The absorbance of the WST formazan dye was measured at 450 nm by using a microplate reader (model 680, Bio-Rad, Hercules, CA, USA) and the glucose amount in each sample was calculated by plotting absorbance values with the standard calibration curve. Experiments were performed with n = 4 replicates.

ADSM were pre-cultured for 16 h in DMEM (Wako, High glucose) without FBS before CM was added directly to a final concentration of 50% and the cells were cultured for another 24 h. The glucose and glutamine concentrations in the medium were measured using a Glucose Assay Kit-WST (DOJINDO, Japan) and Glutamine Assay Kit-WST (DOJINDO), respectively. Absorbance at 450 nm was measured using a FluxStation3 (Molecular Devices) according to the manufacturer’s instructions.

Moisture around the Lymnaea abdomen was thoroughly wiped off with a paper towel, and the tip of a pipette was used to stimulate the mantle. Upon stimulation, 150 µL of hemolymph was collected from the snail’s renal hole. The glucose concentration in the collected hemolymph was measured using a Glucose Assay Kit-WST (Dojindo, Kumamoto, Japan). The working solution for 10 experimental wells was prepared from Dye Mixture Stock Solution:Assay Buffer:Enzyme Stock Solution, which were contained in the kit, at a ratio of 50 µL:450 µL:9 µL. In each well, 50 µL of hemolymph and 50 µL of the working solution were added. After incubation at 37 °C for 30 min, absorbance at 450 nm was measured using a microplate reader (Corona Electronic, Ibaraki, Japan). For calibration, a serial dilution series of 0.5, 0.25, 0.125, 0.0625, 0.0313, 0.0157, 0.00785, and 0 mmol/L of glucose standard was used. The HG snails were compelled to swim in the rearing water (i.e., tap water) for 30 min prior to the collection of hemolymphs to exclude glucose solution on the body surface.