Hydroxyethyl piperazine ethane sulfonic acid hepes

Dulbecco’s Modified Eagle’s Medium (DMEM), calf serum (CS), penicillin, streptomycin, Dimethyl sulfoxide (DMSO), MTT, and DCF-DA were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). A SOD kit was obtained from Dojindo Molecular Technologies (Rockville, MD, USA). Ethanol was purchased from Ethanol Supplies World Co., Ltd. (Jeonju, Republic of Korea). Dimethyl sulfoxide (DMSO), 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), o-phthaldialdehyde (OPT), acetylthiocholine, AChE, pyruvic acid, malic acid, mannitol, egtazic acid (EGTA), hydroxyethyl piperazine ethane sulfonic acid (HEPES), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1), N,O-bis(trimethylsilyl)trifluoroacetamide(BSTFA), and all other reagents used were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).
The primary and secondary antibodies are listed below: ACh, AChE, SYP, PSD-95, GAP-43, TLR-4, NF-κB, IκB-α, IL-1β, TNF-α, BCL-2, BAX, caspase-3, ZO-1, occludin, claudin-1, and β-actin were purchased from Santa Cruz Biotech (Dallas, TX, USA). ChAT and Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody were purchased from Cell Signaling Tech (Danvers, MA, USA). HRP-conjugated anti-mouse IgG secondary antibody purchased from Bio-Rad (Hercules, CA, USA).

Fresh adult bovine knee joints (n = 3) were procured from the butcher for bovine explant research on the day of slaughter, as described previously 8 . Full-thickness articular cartilage explants without the subchondral bone were extracted from the knee joints. Discs of cartilage were then dissected aseptically using a 4 mm dermal punch (Kai Medical, Gifu, Japan). The explants were grown in chondrogenic Eagle's minimal essential medium (DMEM), high (4.5 g/L) glucose (DMEM-HG, Invitrogen, Waltham, USA) supplemented with 1% inulin-transferrin-selenium (Sigma-Aldrich, MA, USA), 1.25 mg/mL bovine serum albumin (Sigma-Aldrich, St. Louis, USA), 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, USA), 0.1 mM ascorbic acid (Sigma-Aldrich, MA, USA), 1% penicillin-streptomycin, 10 mM hydroxyethyl piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, St. Louis, USA), 0.1 mM L-proline (Sigma-Aldrich, St. Louis, USA), 0.1 mM MEM nonessential amino acids (Thermo Fisher Scientific, Waltham, USA) and 10 ng/mL transforming growth factor-β1 (TGF-β1, Gibco, CA, USA)) supplemented with 10% fetal bovine serum (FBS) for 48 h at 37 ℃ and 5% carbon dioxide 22 . The bovine cartilage explants were treated with or without Interleukin-1β (IL-1β) (10 ng/mL) for 7 days to mimic the OA phenotype 23 (link).