Lactate assay kit wst

6.0 × 10⁵ SH-SY5Y cells were cultured in 100 mm dishes and treated with 5, 15, or 30 mM lactate in the differentiation medium, or treated with 10 μM AZD3965 (1448671-31-5, Cayman Chemical) in the growth medium, respectively. Extracellular and intracellular lactate levels were measured by Lactate Assay Kit-WST (L256, DOJINDO) according to the manufacturer’s protocol. Meantime, genome DNA was isolated using Wizard SV Genomic DNA Purification System (A2360, Promega), and the concentration of lactate was normalized to the corresponding genome DNA values.

Cells were washed with cold PBS and lysed in 0.1% Triton X-100. The lysates were filtered using an Amicon Ultra-3 kDa cutoff (Merck). Glucose and lactate levels were quantitated using a Glucose Assay kit-WST and Lactate Assay kit-WST (Dojindo).

The cellular lactate level was measured using the Lactate Assay Kit-WST (Dojindo Molecular Technologies) according to the manufacturer’s protocol. Briefly, 1.0 × 10⁴ cells were seeded in 96-well plates, and the plates were incubated overnight at 37 °C. The medium was exchanged for fresh medium, and the cells were incubated for 24 h. Next, 20 μL of culture supernatant was transferred to a 96-well plate, 80 μL of the working solution was added, and the plates were incubated for 30 min at 37 °C. Finally, the absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific).

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.

Levels of lactate in supernatants were determined by Lactate Assay Kit-WST (DOJINDO, Kumamoto, Japan) according to the manufacturer’s instructions.

Cells were separately cultured as 3 groups; control, 0.3 mM 2-DG treatment, or 1 mM 2-DG treatment groups. On the day before the experiment, cells were washed with PBS and medium was changed to fresh RPMI with appropriate concentration of 2-DG. After 16 h incubation, conditioned medium was collected and used for the assay according to the manufacture’s protocol (Lactate Assay Kit-WST, Dojindo).

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.