Chromatin Immunoprecipitation of Adult Murine Cardiomyocytes

P. Bolli1, C. Vardabasso2, E. Bernstein2, H.W. Chaudhry1

1 Department of Cardiology, Mount Sinai School of Medicine, New York, New York, 2 Departments of Oncological Sciences and Dermatology, Mount Sinai School of Medicine, New York, New York
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.14
DOI:  10.1002/0471143030.cb1714s58
Online Posting Date:  March, 2013
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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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Table of Contents

  • 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 (CaCl 2)
  • 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

Videos

Literature Cited

Literature Cited
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