5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation
Published: May 31, 2023
Abstract
Source: Jia, Z. et al., A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro. J. Vis. Exp. (2017)
This video describes the technique of dot-blot to determine the level of DNA methylation in human chondrocytes in vitro via the blotting of DNA samples on a nylon membrane and the subsequent visualization using monoclonal antibodies. This helps to determine the chondrocytes' phenotype at various stages in culture.
Protocol
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 th…
Disclosures
The authors have nothing to disclose.
Materials
| Reagents | |||
| DMEM | Gibco Inc. | 11965–092 | Warm in 37 °C water bath before use |
| Phosphate-Buffered Saline(PBS) | HyClone Inc. | SH30256.01B | D-PBS, free of Ca2+/Mg2+ |
| FBS | Gibco Inc. | 10099-141 | |
| 0.25%Trypsin/EDTA | Gibco Inc. | 25200-056 | |
| 1%Penicillin-Streptomycin | Gibco Inc. | 15140-122 | |
| Chloroform | Mallinckrodt | 4440 | |
| Isoamyl Alcohol | Sigma | I-3643 | |
| Phenol | Gibco BRL | 15513-039 | |
| Proteinase K | Gibco BRL | 24568-2 | |
| TAE buffer | Bio Whittaker | 16-011V | |
| Distilled Water | Gibco BRL | 15230-170 | |
| 1 M Tris-HCl | Biosharp Inc. | BL514A | |
| Tween20 | Biotopped Inc. | C58H114O26 | |
| BSA | Proliant Inc. | 68700 | |
| Collagenase, Type II | Sigma-Aldrich | C6885 | |
| Equipment | |||
| Cell Strainer (40μm nylon) | FALCON Inc. | 352340 | |
| Hemocytometer | ISOLAB Inc. | 075.03.001 | |
| Falcon 100 mm dish | Corning | 353003 | |
| Centrifuge Tubes | TPP AG | 91050 | Gamma-sterilized |
| High-speed centrifuge | Eppendorf | 5804R | |
| ThermoMixer | MIULAB | MTH-100 | |
| Carbon dioxide cell incubator | Thermo scientific | 3111 | |
| Chemi-imaging Analyse System | UVITEC Cambridge | ALLIANCE |
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Cite This Article
5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation. J. Vis. Exp. (Pending Publication), e21365, doi: (2023).