Cell counting kit 8 cck 8

The following reagents were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany): HAuCl₄·3H₂O, ethanol, Cell Counting Kit-8 (CCK-8), protease inhibitor, DAPI mounting media, and X-treme GENE siRNA transfection reagent. Mouse 3T3-L1 preadipocytes (CL-173, Lot:70047755) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The 3T3-L1 differentiation kit and lipid (Oil Red O) staining kit were obtained from BioVision (Milpitas, CA, USA). The Nile Red staining kit and triglyceride colorimetric assay kit were obtained from Abcam (Cambridge, MA, USA). The following reagents were obtained from Thermo Fisher Scientific Life Sciences (Waltham, MA, USA): Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 (DMEM/F12), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin and streptomycin, eight-well chamber slides, M-PER™ Mammalian Protein Extraction Reagent, Pierce ECL Western blotting substrate, PureLink RNA Mini Kit, high-dose cDNA reverse kit, SYBR Green qPCR Master Mix, PLD1-specific siRNA, and negative control siRNA.

Adherently growing cell lines were seeded at 5,000 cells per 100 μl of cell culture medium in a 96 well flat bottom tissue culture plate (NUNC Inc., VWR International, Ismaning, Germany) and grown for 24 h prior to treatment with chemotherapeutic drugs. Then, 100 μl cell culture medium containing paclitaxel or cisplatin (both from Sigma-Aldrich) were added, resulting in final drug concentrations ranging from 1% to 200% of the reported peak plasma concentrations achievable in patients [49 (link)]. The maximum plasma concentration was given as 15.9 nmol/ml (13.6 μg/ml) for paclitaxel and 12.7 nmol/ml (3.8 μg/ml) for cisplatin [50 (link)]. Cells were incubated for further 24 h and cell viability was measured with Cell Counting Kit-8 (CCK-8; Sigma-Aldrich). In brief, supernatant was carefully removed from the cells and a mixture of 100 μl fresh cell culture medium and 10 μl CCK-8 solution were added per well. Cells were incubated for 1 h at 37°C in a humidified incubator before analyzing the substrate reaction at 450 nm in a plate reader (Tecan GENios plus, Tecan Deutschland GmbH, Crailsheim, Germany). All analyses were performed in triplicates. Results were analyzed with the GraphPad Prism software calculating dose–response curves and IC₅₀ values.

Cells were seeded into 96-well plates and then treated with the following: neutralizing CCL18 antibody (5, 10, 15 or 20 μg/ml), rCCL18 (10, 20, 30 or 40 ng/ml), 20 nmol/l scrRNA, or a combination of 20 nmol/l siCCL18 and 20 ng/ml rCCL18 (siCCL18+rCCL18). Then, cell viability was detected with a Cell Counting Kit-8 (CCK-8) according to the manufacturer's instructions (Sigma-Aldrich, Santa Clara, CA, USA). The absorbance at 450 nm was detected using a microplate reader (Thermo Electron, USA). All experiments were performed in triplicate.