Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Olaf Bergmann; Stefan Jovinge

doi:10.3791/4205

Isolement des noyaux de cardiomyocytes post-mortem des tissus

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Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Isolement des noyaux de cardiomyocytes post-mortem des tissus

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10:12 min

July 10, 2012

DOI: 10.3791/4205-v

Olaf Bergmann¹, Stefan Jovinge^1,2

¹Strategic Research Center for Stem Cell Biology and Cell Therapy,University of Lund, ²Department of Cardiology Lund University Hospital,University of Lund

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Overview

This article describes a method for isolating cardiomyocyte nuclei from postmortem tissue. The process involves homogenization, density sedimentation centrifugation, and fluorescence activated cell sorting to ensure purity.

Key Study Components

Area of Science

Cardiac biology
Nuclear isolation techniques
Flow cytometry

Background

Cardiomyocyte nuclei are critical for studying cardiac function.
Isolation techniques are essential for downstream applications.
Immunolabeling enhances the specificity of nuclear sorting.
Flow cytometry allows for precise sorting based on fluorescence.

Purpose of Study

To isolate cardiomyocyte nuclei for further analysis.
To improve the purity of isolated nuclei for experimental use.
To utilize immunolabeling for enhanced sorting accuracy.

Methods Used

Homogenization of postmortem tissue using a probe homogenizer.
Filtration steps to remove debris and unwanted cellular components.
Density sedimentation centrifugation to isolate nuclear fractions.
Immunolabeling with antibodies against PCM-1 followed by flow cytometry.

Main Results

Successful isolation of cardiomyocyte nuclei from other cellular fractions.
High purity of sorted nuclei confirmed through re-analysis.
Effective use of immunolabeling for precise sorting.
Flow cytometry provided reliable separation of labeled nuclei.

Conclusions

The described method is effective for isolating cardiomyocyte nuclei.
Immunolabeling significantly enhances the sorting process.
High purity of isolated nuclei is achievable, facilitating further research.

Frequently Asked Questions

What is the significance of isolating cardiomyocyte nuclei?

Isolating cardiomyocyte nuclei allows researchers to study cardiac function and gene expression in a controlled manner.

How does immunolabeling improve the isolation process?

Immunolabeling targets specific proteins, enabling more accurate sorting of the desired nuclei.

What role does flow cytometry play in this method?

Flow cytometry allows for the precise separation of labeled nuclei based on fluorescence, ensuring high purity.

Can this method be applied to other cell types?

While this method is optimized for cardiomyocytes, similar techniques can be adapted for other cell types with appropriate antibodies.

What are the potential applications of isolated cardiomyocyte nuclei?

Isolated nuclei can be used for gene expression studies, epigenetic analysis, and other molecular biology applications.

Noyaux cardiaque sont isolés par sédimentation de densité et immunomarquées avec des anticorps contre matériel péricentriolaire 1 (PCM-1) d'identifier et de trier les noyaux des cardiomyocytes par cytométrie de flux.

L’objectif global de cette procédure est d’isoler les noyaux des cardiomyocytes du tissu post-mortem. Pour ce faire, il suffit d’homogénéiser d’abord le tissu à l’aide d’une sonde homogénéisatrice et d’un verre downer, puis de procéder à plusieurs étapes de filtration. La deuxième étape consiste à isoler la fraction nucléaire des autres fractions cellulaires par centrifugation par sédimentation densité.

Ensuite, les noyaux des cardiomyocytes sont immunomarqués avec des anticorps dérivés contre la PCM un. La dernière étape consiste à isoler les noyaux de cardiomyocytes marqués par tri cellulaire activé par fluorescence. En fin de compte, juste avant la manipulation ultérieure en aval, la pureté des noyaux de cardiomyocytes est confirmée par une réanalyse des fractions nucléaires triées.

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