Cck 8 cell viability kit

Primary glomerular microvascular endothelial cells (GMVEC), purchased from Cell Systems (Kirkland, WA, USA), were cultured in complete classic medium (#4Z0-500) supplemented with culture boost. The passage number was used between 3 to 7. This primary cell line was utilised as any other relevant cell lines are immortalised rather than human primary cells and were therefore not appropriate. Endothelial growth factor (VEGF-A₁₆₅) was purchased from Thermo-Fisher (Paisley, UK). Pure and analytical grade artificial sweeteners, aspartame, saccharin, and sucralose, the CCK-8 cell viability kit, FITC-dextran, N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), methanol, and ethyl acetate were purchased from Sigma-Aldrich (Dorset, UK). Lactisole, a sweet taste inhibitor, was purchased from Cayman Chemical (Ann Arbor, MI, USA). The cAMP-Screen Direct System kit and GSH Bioxytech activity kit were purchased from Applied Biosystems and Merck Millipore respectively. DharmaFECT™ reagent and siRNA (T1R3 and non-specific, scrambled) were purchased from Dharmacon (Cambridge, UK). Anti VE-cadherin and fluorescent secondary antibodies, deuterated sucralose, and sucralose-D6 (SC-220145) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2,7-dichlorodihydrofluorescein diacetate (DCFDA) was purchased from Abcam (Cambridge, UK).

BV2 cells were seeded into 96-well plates at a density of 10⁴ cell/well and cultured for 24 h. After LPS and LTA ± IL-6 neutralizing antibody treatments, cell viability was determined using the CCK-8 Cell Viability Kit (Sigma-Aldrich Kft.) according to the manufacturer’s protocol. Briefly, 10 µL of WST-8 reagent was added to each well, and then the plates were incubated for 1 h at 37 °C and 5% CO₂. The reaction was stopped by adding 10 µL of 1% SDS solution to the cells. OD values of the samples were measured at 450 nm using MultiSkanGO Microplate Reader (Thermo Fisher Scientific Inc., Waltham, MA, USA) and cell viability was calculated as percentile of the cell number of the control cells.