Penicillin streptomycin antibiotics p s

The knee cartilage of C57BL/6 mouse was isolated and then digested using 5‐10 mL of 2 mg/mL type‐II collagenase (Sigma‐Aldrich) at 37°C for 4 hours. After centrifugation, the supernatant was inoculated in a petri‐dish with DMEM/F12 (Gibco) contained 10% foetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) antibiotics (Gibco). The chondrocytes were then cultured at 37°C with 5% CO₂. The cells of passage# 2 were used for subsequent experiments.

The chemicals, COX2 enzymes and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) reagent, were acquired from Sigma-Aldrich Corp. in St. Louis, MO, USA (cat no. 1898-66-4). A centrifugal ultrafiltration filter YM-30 (cat no. 42410) from Millipore Co., Ltd., Burlington, Middlesex County, MA, USA. with a 30 kDa capacity was bought. All other chemicals and solvents were of analytical grade and were obtained from Duksan Pure Chemical Co., Ltd. (Dongdaemun-gu, Seoul, Republic of Korea). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), and penicillin/streptomycin (P/S) antibiotics were acquired from Gibco (BRL Life Technologies, Grand Island, NY, USA). COX2 (cat. no. 12282S), iNOS (cat. no. 13120S), and β-actin (cat. no. 3700S) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies against rabbit and mouse were purchased from Bethyl Laboratories, Inc. (Montgomery, AL, USA) using horseradish peroxidase (HRP) conjugation.

MP41 cell line was obtained from Decaudin’s laboratory at Curie Institute, Paris, France. Mel202 and 92.1 cell lines were obtained from European Collection of Authenticated Cell Cultures (ECACC) (Merck). 501Mel cell line was obtained from American Type Culture Collection (ATCC). HCT116 WT and HCT116 KO DROSHA (Kim et al, 2016b (link)) cell lines were obtained from Korean Collection for Type Cultures (KCTC), Microbial Resource Center. All cell lines were maintained in humidified air (37°C, 5% CO₂). UM cell lines were maintained in RPMI-1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 20% FBS (EurobioScientific) and 1% penicillin–streptomycin antibiotics (Gibco, Thermo Fisher Scientific). 501Mel was maintained in RPMI-1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (EurobioScientific) and 1% penicillin–streptomycin (PS) antibiotics (Gibco, Thermo Fisher Scientific). HCT116 cell lines were maintained in McCoy’s 5A (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (EurobioScientific) and 1% penicillin–streptomycin antibiotics (Gibco, Thermo Fisher Scientific). All cell lines have been routinely tested for mycoplasma contamination (Mycoplasma contamination detection kit; rep-pt1; InvivoGen).

Dulbecco’s modified Eagle medium (DMEM) and penicillin/streptomycin antibiotics (P/S) were purchased from Gibco (Gaithersburg, MD, USA); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent and corticosterone were purchased from Sigma-Aldrich (St. Louis, MO, USA). Serotonin, dopamine, interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) ELISA kits were procured from Abcam (Cambridge, MA, USA).

The American Type Culture Collection (ATCC) mouse embryonic myoblast C2C12 cells, as precursors of skeletal muscle cells (passage 8-13) were seeded in a culture dish at a density of 2 × 10³ cells/cm². The control group(G1) was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco® Life Technologies, USA) and 10% (v/v) fetal bovine serum (FBS; WELGENE, Korea) as a standard growth medium (GM) with 1% penicillin/streptomycin antibiotics (PS; Gibco® Life Technologies, USA). The experimental groups (G2 and G3) were cultured in DMEM with 5% FBS (G2) or 5% FBS + 50 μg/ml C -phycocyanin (C-PC, Sigma Aldrich) (G3), each with 1% PS. All three groups were cultured at 37 °C under a humidified atmosphere containing 5% CO₂ and incubated for 7 days. The GM was replaced every 2 days. Upon reaching 100% confluence (i.e., after 7 days), the culture medium was replaced with a differentiation medium (DM) consisting of 5% horse serum (HS; Thermo Fisher Scientific) and 1% PS, and the cells were differentiated for 5 days. During this time, the DM was replaced every 2 days.

Dulbecco’s Modified Eagle Medium (DMEM), phosphate-buffered saline (PBS), and penicillin-streptomycin antibiotics (P/S) were supplied by Gibco (Gaithersburg, MD, USA). Fetal bovine serum (FBS) was obtained from GenDEPOT (Barker, TX, USA). Primary antibodies and secondary antibodies were purchased from Abcam (Cambridge, MA, USA). Lipopolysaccharide (LPS), ovalbumin (OVA) from chicken egg, aluminum hydroxide (Alum), anti-dinitrophenyl (DNP) IgE, DNP-human serum albumin (HSA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).