Cells were cultured in DMEM/F12 (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% Selleckchem BIBF1120 antibiotic (100 U/ml penicillin and 0.1 mg/ml streptomycin, Sigma-Aldrich Corporation, St. Louis, MO, USA) in an incubator (5% CO2, 37°C). The medium was refreshed every 3 days, and
cells were split 1:3 after reaching 90% confluence. Chondrogenic differentiation ADSCs (passage 3) were seeded at a high-cell density (2 × 105/10 ml), then the medium was changed to DMEM/F12 supplemented with chondrogenic Selleck GSK2245840 medium: 1% FBS, 6.25 μg/ml insulin + ITS (Sigma, USA), 10 ng/ml TGF-β1 (Peprotech, Rocky Hill, NJ, USA), 10 to 7 M dexamethasone (Sigma, USA), 50 μg/ml ascorbic acid (Sigma, USA), 100 U/ml penicillin, and 0.1 mg/ml
streptomycin as previously described . Twenty-one days after induction, lipid accumulations in adipocytes were visualized by staining with oil red-O as follows: cells were fixed in 10% formalin for 1 h Rabusertib mouse and stained for lipid with 0.3% oil red-O for 15 min. After rinsing three times with double distilled H2O, the red-staining cells in six random areas of 1 mm2 were counted in each well and presented as an average ± standard deviation for 3 to 6 replicate wells. Chondrocytes isolation and culture Cartilage was obtained from six patients (mean age, 58 years; range, 40 ~ 78 years) undergoing total hip replacement at the First Affiliated Hospital of Jinan University, learn more with femoral neck fracture. Chondrocytes were isolated and collected according to the procedure proposed
by Malicev et al. , with slight modifications. Culture medium contains DMEM/F12 supplement with 10% FBS. Primer design The primers for amplification of Aggrecan, COLII, SOX9, and COLI were designed using Primer Express 5.0 software using default parameters according to the published sequences in Gen-Bank. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a positive control. The primer sequences are listed in Table 1. All primers were obtained from Invitrogen. Table 1 Sequences of primers for real-time PCR Primer name Forward primer (5′-3′) Reverse primer (5′-3′) Product size (bp) Aggrecan 5 ′ -CTGCCCCAGAAGTGAGTGGAG-3 ′ 5 ′ -TGGTGCTGATGACAACGCCC-3 ′ 159 COL II 5 ′ -CACCTGCAGAGACCTGAAA-3 ′ 5 ′ -CAAGTCTCGCCAGTCTCCAT-3 ′ 126 Sox-9 5 ′ -AACGCCATCTTCAAGGCG-3 ′ 5 ′ -CTCTCGCTTCAGGTCAGCCTT-3 ′ 165 COL I 5 ′ -CCTGGATGCCATCAAAGTCT-3 ′ 5 ′ -ACTGCAACTGGAATCCATCG-3 ′ 150 GAPDH 5 ′ -CCACCATGGAGAAGGCTG-3 ′ 5 ′ -GGTGCTAAGCAGTTGGTCCT-3 ′ 170 RNA isolation and real-time-polymerase chain reaction analysis Total RNA was extracted using Trizol (Invitrogen, USA) protocol. Two micrograms of total RNA was used for reverse transcription reaction with the RevertAid First Strand cDNA synthesis kit (Fermentas, Thermo Fisher Scientific Waltham, MA, USA) and random oligo(dT) primer (Fermentas), according to the manufacturer’s instructions.