Cls 2

This research was approved by the Ethics Committee of Tianjin Hospital, China (2014–008). Discarded cartilage tissues were obtained from 10 normal patients (without OA) undergoing traumatic above-knee amputation and 10 OA patients undergoing total knee replacement surgery, aged 47–78 years. The OA patients were clinically diagnosed to be Kellgren-Lawrence grade 3 based on radiographic examination. All clinical specimens were obtained after patients gave informed consent. The two groups were paired and cartilage samples were matched by age, sex and body mass index.
Chondrocytes were isolated from the articular cartilage of clinical specimens obtained from normal and OA patients. Cartilage samples were minced and digested in 0.15% (w/v) collagenase (CLS-2, Worthington, USA) for 16 h at 37 °C, in medium consisting of Dulbecco’s Modified Eagle Medium (DMEM, Gibco, UK) supplemented with 10% fetal bovine serum (FBS, HyClone, USA), 100 U/mL penicillin (Gibco) and 100 μg/mL streptomycin (Gibco). Isolated chondrocytes were washed in PBS and filtered through a 100 μm cell strainer (BD Biosciences, USA). The cells were seeded at high density (1 × 10⁴ cells/cm²) and kept in maintenance medium for 2 days prior to gene expression analysis.

Cardiomyocytes were isolated from left ventricular tissue using a method similar to Shioya (2007) (link). Hearts were rapidly removed from mice anesthetized with an overdose of pentobarbital (300 mg/kg, i.p.). Isolated hearts were perfused with cell-isolation buffer (CIB; 130 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl₂, 0.33 mM NaH₂PO₄, 22 mM glucose, 50 nM/mL bovine insulin (I6634, Sigma) and 25 HEPES-NaOH (pH 7.4)) supplemented with 0.4 mM EGTA at 37°C. After 3-4 min, the perfusate was changed to the enzyme solution, which was CIB containing 0.3 mM CaCl₂, 1 mg/mL collagenase (CLS-2, Worthington Biochemical), 0.06 mg/mL protease (P5147, Sigma) and 0.06 mg/mL trypsin (T8003, Sigma). After 6-9 mi, the tissue was cut into several pieces and further digested in fresh enzyme solution containing 0.7 mM CaCl₂ and 2 mg/mL BSA (A9418, Sigma) for 15–20 min at 37°C. After the cell suspension was centrifuged at 14 × g for 3 min, the cell pellet was resuspended in CIB containing 1.2 mM CaCl₂ and 2 mg/mL BSA, and then incubated for 10 min at 37°C. The cell suspension was centrifuged (14 × g, 3 min) again and resuspended in Tyrode’s solution (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 0.33 mM NaH₂PO₄, 11 mM glucose, 2 mg/mL BSA and 5 mM HEPES-NaOH (pH 7.4). Experiments of isolated cardiomyocytes were performed with cells superfused with Tyrode’s solution unless otherwise indicated.

We digested cardiac tissue from a subset of rats in the hypoxic and normoxic groups to obtain a single‐cell suspension for flow cytometry. Rats were anesthetized as indicated above, the heart was flushed with PBS, and the RV and LV free walls were separated. Tissue was cut into ~1 mm³ pieces and placed in PBS with collagenase II (150 U/ml, Worthington Biochemical Co., CLS‐2) and incubated for 1 hr at 37°C. Tissue was then triturated and filtered through a 40 μm filter and centrifuged at 250g for 8 min. The resulting pellet was resuspended in FACS buffer (PBS with 0.5% BSA and 1 mM EDTA) and stained with antibodies directed against rat CD45‐FITC (Biolegend 202205), CD11b/c‐APC (Biolegend 201809) (leukocyte surface markers) or the respective isotype controls (Biolegend 400107 and 400219) for 20 min, followed by washing with FACS buffer. Single‐stain groups were used for compensation, a method that corrects for the spectral overlap between the different emission spectra of fluorochromes. We conducted flow cytometry in each sample on at least 50,000 events using the BD FACSCanto II (BD Biosciences). Data were analyzed using FlowLogic (Miltenyi Biotec).