Cck 8

Cells were seeded into 96-well plates (2×10⁴ cells/well). After exposure to experimental conditions for 24 hours, cell viability was measured by CCK-8 (Dojindo Molecular Technologies) per manufacturer’s instructions. Briefly, CCK-8 utilizes a water-soluble tetrazolium salt WST-8. On reduction by dehydrogenases in cells, a yellow coloured formazan product is formed, which is proportional to the number of living cells. The CCK-8 results were consistent with the trypan blue exclusion assay in our studies (data not shown).
Measurement of sFLT-1 and sENG in Culture Supernatants sFLT-1 and sENG in supernatants was measured by Quantikine ELISA assays (R&D Systems, Minneapolis, Minnesota, USA) per manufacturer’s instructions. The interassay coefficients of variation (CVs) were 10% and 16%, respectively, and intra-assay CVs were 9% and 8%, respectively. Data were presented as ‘cell viability adjusted’, that is, the biomarker of interest (ng/L)/proportion of alive cells.

A CCK-8 (R&D Systems, Minneapolis, MN, United States) assay was adopted to detect betaine (RHAWN, R20086) cytotoxicity following the manufacturer’s protocol. In brief, BMMCs at a concentration of 10 × 10⁴ cells/ml were added to 96-well plates. After 24 h, multiple concentrations of betaine (0, 31.25, 62.5, 125, 250, 500, 1,000 , 2000 μM) were added to the wells and incubated for another 48 h. Then, CCK-8 solution (10 μl) was added and cocultured for 2 h in an incubator. The absorbance level of each well at 450 nm was determined by an ELISA microplate reader.