4 2 hydroxyethyl 1 piperazineethane sulphonic acid

Cortical and hippocampal neuron cultures were prepared from E17.5 mouse embryos (44 (link)). Briefly, embryos were individually decapitated and cortex or hippocampi were dissected and collected in cold Hank’s balanced salt solution (HBSS: NaHCO3 4.2 mm, Hank’s salt powder 0.952%, HEPES 12 mm, 4-(2-hydroxyethyl)-1-piperazineethane-sulphonic acid, Sigma), then digested with 0.25% Trypsin at 37°C for 8 min. Enzymatic digestion was blocked with Dulbecco’s Modified Eagle Medium high glucose (DMEM), supplemented with 10% Fetal Bovine Serum (FBS) and penicillin-streptomycin. The tissue was centrifuged at 800 rpm for 5 min at room temperature. The tissue was resuspended with DMEM + 10% FBS and mechanically triturated. Hippocampal neurons were plated on 12 mm cover glasses and cortical neurons directly on polystyrene multiwell plates, coated with 1 mg/ml poly-L-lysine (Sigma). Cultures were grown at 37°C and 5% CO₂ in Neurobasal Medium (Invitrogen) supplemented with 2% B-27 (Invitrogen), 1 mm L-glutamine and 1% penicillin-streptomycin. Genotype and sex were later confirmed by PCR.

Sprague Dawley neonatal rats were obtained from the Animal Center of Xinxiang Medical University (Xinxiang, China). The study and animal use were approved by the Ethics Committee of Xinxiang Medical University. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco-BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) and a cell counting kit-8 (CCK-8) were purchased from Hangzhou Sijiqing Bioengineering Material Co., Ltd. (Hangzhou, China). Enhanced green fluorescent protein (EGFP)-adenovirus capsids with and without CyclinA2 cDNA were obtained from Shanghai Genechem Co., Ltd. (Shanghai, China). Rabbit anti-rat CyclinA2 and mouse anti-rat β-actin primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Total protein extraction and bicinchoninic acid (BCA) protein analysis kits were purchased from Pierce (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Polyvinylidene fluoride (PVDF) membranes, trypsin, 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid, tetramethylethylenediamine and EDTA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Centrifugal filter units were purchased from Millipore Corporation (Billerica, MA, USA).

Tryptic Soy Broth (TSB), deMan, Rogosa, and Sharpe (MRS) Broth and agar, fructose, cysteine-hydrochloride, sodium chloride (NaCl), sodium hydroxide (NaOH), phosphate-buffered saline (PBS), high-glucose Dulbecco’s Modified Eagle Medium (DMEM), trypsin, 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), methanol, streptomycin–penicillin mixture for cell cultures, trypan blue, mucin from the porcine stomach, acetic acid, ethanol, gelatin, crystal violet, Triton X-100, and n-hexadecane were purchased from Merck Life Science, Warsaw, Poland. Anaerobe Basal Broth (ABB), foetal bovine serum (FBS), GlutaMAX^TM, TrypLE^TM Express, and AnaeroGen^TM Atmosphere Generation Systems sachets were purchased from Thermo Fisher Scientific, Waltham, MA, USA. Cryobanks™ were from Copan Diagnostics Inc., Jefferson Avenue Murrieta, Murrieta, CA, USA. In addition, 6-, 24- and 96-well transparent flat-bottom plates, serological pipettes, and T75 roux flasks (all from Greiner Bio-One GmbH Kremsmünster, Austria) were purchased in Biokom Systems, Janki, Poland. Collagen-coated 96-well plates (BioCoat^®®) were from Becton, Dickinson and Co., Franklin Lakes, NJ, USA. Disposable syringe filters (0.22 µm pore size) were purchased from Labindex S.A., Warsaw, Poland. The Caco-2 cell line was from Cell Line Service GmbH, Eppelheim, Germany.