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).

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