Dmem f12k

In this work, the effect of the pre-treatment of bMECs with γ-thionin was analyzed with or without the infection with S. aureus. Prior to the infection, bMECs were incubated with γ-thionin (100 ng/ml) in DMEM/F12K (Sigma) without antibiotics and serum for 24 h. The infection assays were performed using gentamicin protection assays as described in the following section, and bMECs were infected for 2 h (10 (link)). bMECs treated with vehicle (DMSO 0.02%) were employed as control in all of the experiments. For the RNA, nuclear protein, or total protein extractions, bMECs were processed according to the protocols described below. For flow cytometry analysis of MAPK activation, total protein was obtained from cell lysates, and for AKT analysis, flow cytometry was performed in whole cells (see below). Enzymatic activity of HDACs was determined in cultured bMECs, whereas HDM activity was measured in cell lysates.

For enrichment of sphere-forming cells, parent cancer cells were cultured at the indicated density on Costar Ultra-Low Attachment plates (Corning, Corning, NY, USA) or six-well plates coated with poly (2-hydroxyethyl methacrylate) (polyHEMA; Sigma-Aldrich Co.) in defined medium consisting of serum-free DMEM/F12-K, 1x ITS solution (Sigma-Aldrich Co.), 20 ng/mL human recombinant EGF, and 20 ng/mL human recombinant bFGF. polyHEMA-coated plates were prepared by adding 1 mL of a 10 mg/mL polyHEMA solution in 95% ethanol to the six-well plates and incubating overnight in a laminar flow hood at room temperature for air drying.

Fetal bovine serum, penicillin–streptomycin, Lysotracker Red-DND, CalceinAM were purchased from Thermo Fisher Scientific (Eugene, OR, USA). DMEM F-12K, Neutral red dye, MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], Cacodylic acid, Pyrogallol, Bradford reagent, DCFH-DA, JC-1, H₂O₂, TBA, NADPH, pyruvic acid, H₂O₂ measuring kit, Hoechst, PI, all NPs were obtained from Sigma–Aldrich, USA. Ultrapure deionized-water was prepared using a Milli-Q system (Millipore, Bedford, MA, USA). All other chemicals used were of reagent grade.