The overall goal of this experiment is to determine the 5-Methylcytosine levels during chondrocyte dedifferentiation. This method shows can help answer key questions in cell-based cartilage defect treatment such as autologous chondrocyte implantation. The main advantage of this technique is that it is a reliable, simple and rapid method for detecting the general DNA methylation level to evalute chondrocyte phenotype.
The implication of this technology is standard to our share of autologous chondrocyte implantation because DNA methylation during chondrocyte dedifferentiation is a major obstacle for autologous chondrocyte implantation treatment for cartilage defects. Though this method can provide insight into DNA methylation levels in chondrocyte dedifferentiation, it can also be applied to other studies of diseases, such osteoarthritis. We first had the idea for this method when we read the protocols in the references of a dot blot method.
Please show demonstration of this method is critical as a dot blot states are difficult to name. Genomic DNA should be denatured and then be serially diluted. Then be supported on a positively-charged nylon membrane.
Demonstrating the procedures will be Zhaofeng Jia, a graduate student from our lab. The articular cartilage tissues used in this study were isolated from the knee joints of donor patients after trauma. Using a sterile scalpel, dice the cartilage into one to two cubic millimeter pieces.
Then add one milligram per milliliter of collagenase II and DMEM to the minced cartilage. And incubate at 37 degrees Celsius for 12 to 16 hours to digest the chondrocytes. On the following day, filter the resulting cell suspension through a 40 micrometer cell strainer.
Spin down the cells. Discard the supernatant, add PBS, and spin down again. Count the cells with a hemocytometer.
And seed at a density of 20, 000 to 30, 000 cells per centimeter squared and DMEM supplemented with 10%FBS and 1%penicillin streptomycin. Culture in an incubator at 37 degrees Celsius for 48 hours. Harvest subconfluent cells using 0.25%trypsin EDTA.
And replate at a density of 6, 600 cells per centimeter squared. Culture the chondrocytes in monolayers for up to six passages and assess at passages one, two, three, four, and five. To begin this procedure, collect cells by trypsinization and centrifugation.
Then resuspend cells in 500 microliters of lysis buffer per 10 million cells by pipetting and rapid inversion. Incubate for one hour at 37 degrees Celsius. Add proteinase K at a concentration of 160 micrograms per milliliter of cell lysate and invert the mixture vigorously.
Incubate for six hours at 55 degrees Celsius. Add to each sample one volume of Tris pH 7.9 saturated phenol to chloroform to isoamyl alcohol. Shake the sample by hand thoroughly for approximately 20 seconds.
Centrifuge the samples at room temperature at 13, 000 times G for five minutes. Remove the upper aqueous phase, and transfer to a fresh tube. To remove the phenol, add an equal volume of chloroform to each tube.
Shake by hand, and spin down. Transfer the top aqueous phase to a fresh tube. Add 0.1 sample volume of three molar sodium acetate pH eight and two volumes of 100%ethanol.
Store the tubes at negative 20 degrees Celsius overnight to precipitate the genomic DNA. On the following day, centrifuge the samples at four degrees Celsius for 10 minutes at 16, 000 times G to pellet the genomic DNA. Wash the samples with 70%ethanol.
And centrifuge at four degrees Celsius for two minutes at 13, 000 times G.Remove the supernatant carefully and air dry the DNA pellet. Resuspend the DNA in 10 millimolar tris pH eight 0.1 millimolar EDTA. Start this procedure by denaturing the isolated DNA in 0.1 molar sodium hydroxide for 10 minutes at 95 degrees Celsius.
Neutralize the DNA with one molar ammonium acetate on ice. And then dilute twofold with double distilled water. The most critical step in the dot blot assays is the spotting of the samples, and remember do it slowly and carefully.
Try to minimize each spotted array to three to four millimeter in diameter. Using a narrow mouth pipette tip, carefully spot two microliters of the serial diluted genomic DNA onto a positively charged nylon membrane at the center of the grid. Blot the membrane at 80 degrees Celsius for 30 minutes.
Next, block the non-specific antibody binding sites by soaking the membrane in 5%BSA in TBST in a 10 centimeter petri dish for one hour at room temperature with gentle shaking. After one hour, wash the membrane three times in TBST for five minutes each time. Incubate the membrane with a mouse anti-5-methylcytosine monoclonal antibody in TBST at four degrees Celsius overnight.
On the following day, wash the membrane three times in TBST for five minutes each time. Then incubate with a secondary antibody, horseradish peroxidase conjugated sheep anti-mouse immunoglobulin G in TBST for one hour at room temperature. After washing the membrane in TBST as before, add the enzyme substrate to the membrane and incubate for five to 10 minutes.
Finally, visualize the secondary antibody signal using a chemiluminescence kit according to the manufacturer's instructions. Chondrocytes cultured in a monolayer up to passage six showed progressive phenotypic changes becoming highly pinched and flattened with successive passages. This morphology change is typical of the chondrocyte dedifferentiation process.
Genomic DNA was isolated from chondrocytes after varying number of passages and DNA methylation levels were assessed by a 5-Methylcytosine specific dot blot assay. Intensities of the dots were then analyzed by image J.Progressively elevated five methylcytosine levels were observed with prolonged subculture, suggesting an association between generally increased methylation levels and chondrocyte dedifferentiation. Once mastered, this technique can be done in 24 hours if it is performed properly.
While attempting this procedure, it's important to remember to maximize genomic DNA concentration and purity. Following this procedure other methods like genomic DNA machination profiling can be performed to answer additional questions such as chondrocytes of dedifferentiation in monoculture. After its development, this technique paved the way for researchers in the field of autologous chondrocyte implantation to evaluate DNA machination of chondrocytes and determine the chondrocyte phenotype in cartilage defect treatment.
After watching this video, you should have a good understanding of how to use 5-Methylcytosine dot blot to detect degenerate machination lever to evaluate the chondrocyte phenotype. Don't forget that working with phenol can be extremely hazardous and gloves should always be worn while performing this procedure.
