Chromatin Immunoprecipitation of Adult Murine Cardiomyocytes

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes a streamlined two?step protocol for the isolation of adult murine cardiomyocytes with subsequent Chromatin ImmunoPrecipitation (ChIP). Isolation and culturing of cardiomyocytes is a delicate process and the protocol presented here optimizes the combination of cardiomyocyte isolation with ChIP. ChIP is an invaluable method for analyzing molecular interactions occurring between a specific protein (or its post?translationally modified form) and a region of genomic DNA. ChIP has become a widely used technique in the last decade since several groundbreaking studies have focused attention on epigenetics and have identified many epigenetic regulatory mechanisms. However, epigenetics within cardiovascular biology is a new area of focus for many investigators, and we have optimized a method for performing ChIP in adult murine cardiomyocytes, as we feel this will be an important aid to both the cardiovascular field and for the development of cell? and tissue?specific ChIP. Curr. Protoc. Cell Biol. 58:17.14.1?17.14.16. © 2013 by John Wiley & Sons, Inc.

Keywords: Chromatin ImmunoPrecipitation; adult murine cardiomyocytes; cardiomyocyte isolation

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Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1:

Materials

Perfusion buffer (see recipe )
Digestion buffer (see recipe )
Stop buffer 1 (see recipe )
Stop buffer 2 (see recipe )
Cardiomyocyte medium (see recipe )
Isoflurane
Adult mouse (the type of mouse used will depend on the aim of the experiment)
70% ethanol
Collagenase D (Roche)
2,3 butanedione monoxime (BDM; Sigma)
Calcium chloride (CaCl₂ )
Liquid nitrogen
Ice
Phosphate‐buffered saline (PBS; Mediatech, cat. no. 21‐030)
Protease inhibitor cocktail (Roche, cat. no. 118361770001)
16% formaldehyde solution (Electron Microscopy Sciences) or 18.5% formaldehyde solution (see recipe )
Glycine (see recipe )
Cell lysis buffer (see recipe )
Nuclei lysis buffer (see recipe )
RNase, optional
Proteinase K, optional
Agarose gel, optional
Gel loading buffer, optional
Gel loading dye, optional
100‐bp DNA marker, optional
Dilution buffer (see recipe )
Antibodies to protein of interest (preferentially ChIP‐grade)
Antibody (Normal rabbit IgG)
Protein A magnetic beads (alternatively Protein G magnetic beads)
Low salt wash buffer (see recipe )
High salt wash buffer (see recipe )
LiCl wash buffer (see recipe )
TE buffer (see recipe)
Elution buffer (see recipe )
Molecular grade water (Mediatech, cat. no. 46‐000)
Primers specific for analysis of the regions of interest
2× Sybr green (Roche, cat. no. 04673484001)

37°C water bath
Perfusion apparatus: Langendorff apparatus
Surgical apparatus (e.g., prepare a surgical table by wrapping a polystyrene lid with aluminum foil)
Gel loading tips
Blade
22‐G feeding needle, straight, with a ball tip, optional
100‐mm petri dishes
Braided silk suture (6‐0, metric 0.7)
Dissecting microscope
1‐ml syringes
Surgical table
Surgical instruments (all surgical tools are from Fine Science Tools) including:
- Dumont #5 fine‐tip forceps for the aorta cannulation
- Small scissors
Laminar flow culture hood
1000‐µl pipet tips
100‐µm filters
15‐ and 50‐ml conical tubes
Centrifuge
Hemacytometer
Ice bucket
Rotating platform, at 4°C and at room temperature
Vortex mixer
Sonicator (e.g., Bioruptor from Diagenode)
1.5‐ml microcentrifuge tubes
Heating block preset to 95°C
Shacking incubator, preset at 62°C
Gel electrophoresis apparatus, optional
Magnetic separation rack
DNA purification kit (e.g., Qiagen)
Thermal cycler

Additional reagents and equipment for animal euthanasia (Donovan and Brown, )

CAUTION: Formaldehyde is a biohazard. Consult MSDS for proper handling instruction.NOTE: Any protocol using vertebrate animals must be approved by an Institutional Animal Care and Use Committee (IACUC) and must follow approved procedures for the care and use of laboratory animals.

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Figures

Figure 17.14.1 Schematic of the protocol.

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Figure 17.14.2 Cannulation setup.

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Figure 17.14.3 Diagrammatic illustration of the different steps to be performed during heart perfusion.

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Figure 17.14.4 Agarose gel analysis of sonicated DNA.

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Figure 17.14.5 Data analysis with (A ) percent input method and (B ) fold enrichment method.

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Videos

Literature Cited

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