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1. Human Articular Cartilage Tissue Collection and Chondrocyte Culture
- Preparation of materials
- Prepare Dulbecco's modified eagle medium (DMEM) medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Prepare 1 mg/mL collagenase II, 0.25% trypsin-EDTA, phosphate-buffered saline (PBS), and a cell strainer (40 µm nylon).
- Human articular cartilage tissue collection
- Isolate articular cartilage from the knee joints of donor patients after trauma. Obtain informed consent from all participants.
- Dice the cartilage into 1-2 mm3 pieces using a sterile scalpel and digest chondrocytes from the minced cartilage with 1 mg/mL collagenase II in DMEM at 37 °C for 12-16 h.
- Filter the resulting cell suspension through a cell strainer (40 µm) and wash twice with PBS.
- Count cells with a hemocytometer and seed at a density of 20,000-30,000 cells/cm2 in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.
- Culture in an incubator at 37 °C.
- Monolayer chondrocyte expansion
- Harvest sub-confluent cells using 0.25% trypsin-EDTA and re-plate at a density of 6,600 cells/cm2. Change the medium twice a week.
- Culture chondrocytes in monolayers for up to six passages and assess at passages 1, 2, 3, 4, and 5.
2. Genomic DNA (gDNA) Extraction
- Collect cells and resuspend in 500 µL lysis buffer (15 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 0.5% SDS, 200 µg/mL RNase A) per 10 million cells. Resuspend cells by pipetting and rapid inversion, and then incubate for 1 h at 37 °C.
- Add proteinase K at a concentration of 160 µg/mL of cell lysate and invert the mixture vigorously. Incubate 6 h at 55 °C.
- Add one volume of Tris pH 7.9 saturated phenol:chloroform:isoamyl alcohol (25:24:1) to the sample. Vortex or shake the sample by hand thoroughly for approximately 20 s.
- Centrifuge the sample at room temperature for 5 min at 13,000 × g. Remove the upper aqueous phase and transfer the layer to a fresh tube.
- Extract with an equal volume of chloroform to remove phenol and transfer the top aqueous phase to a fresh tube.
- Precipitate DNA by adding 0.1 sample volume of 3 M sodium acetate pH 8.0 and 2 volumes of 100% ethanol.
- Store the tube at -20 °C overnight to precipitate the gDNA.
- Centrifuge the sample at 4 °C for 10 min at 16,000 x g to pellet gDNA.
- Wash the sample three times with 70% ethanol, and centrifuge at 4 °C for 2 min at 13,000 x g.
- Remove the supernatant carefully and then air dry. Resuspend the DNA in 10 mM Tris pH 8.0, 0.1 mM EDTA.
3. DNA Methylation Profiling
- Use a DNA methylation kit to perform the bisulfite conversion reaction using a total of 500 ng of the genomic DNA, according to the manufacturer's protocol. Elute in 10 µL of elution buffer (50 ng/µL).
- Perform the DNA methylation profiling using a commercial kit according to the manufacturer's protocol.
4. Dot Blot Analysis
- Denature the isolated DNA (1 mg per sample) in 0.1 M NaOH for 10 min at 95 °C. Neutralize the DNA with 1 M NH4OAc on ice, and then dilute two-fold. Spot 2 µL of the serial diluted genomic DNA on an N+ membrane.
- Blot the membrane at 80 °C for 30 min.
- Block non-specific antibody binding sites by soaking the N+ membrane in 5% BSA in TBS-T for 1 h. Use a 10 cm Petri dish as a reaction chamber at room temperature.
- After washing 5 min three times in TBST, incubate the membrane with a mouse anti-5-methylcytosine (5-mC) monoclonal antibody (1:1,000) in TBS-T at 4 °C overnight.
- Wash the membrane for 5 min three times in TBS-T, and then incubate with a secondary antibody, HRP-conjugated sheep anti-mouse immunoglobulin-G (IgG) (1:5,000) in TBS-T for 1 h at room temperature.
- Wash the membrane for 5 min three times in TBS-T.
- Add the enzyme substrate to the membrane and incubate for 5-10 min. Visualize the secondary antibody signal using a chemiluminescence kit according to the manufacturer's instructions.