5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

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

1. Preparation of materials
   1. 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).
2. Human articular cartilage tissue collection
   1. Isolate articular cartilage from the knee joints of donor patients after trauma. Obtain informed consent from all participants.
   2. Dice the cartilage into 1-2 mm³ pi....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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