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Biology
Isolement de cellules endothéliales glomérulaires de souris immortalisées conditionnellement avec...
Isolement de cellules endothéliales glomérulaires de souris immortalisées conditionnellement avec...
JoVE Journal
Biology
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JoVE Journal Biology
Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Isolement de cellules endothéliales glomérulaires de souris immortalisées conditionnellement avec des mitochondries fluorescentes

Full Text
2,672 Views
11:49 min
September 13, 2022

DOI: 10.3791/64147-v

Rihab Bouchareb1, Liping Yu1, Emelie Lassen1, Ilse S. Daehn1

1Department of Medicine, Division of Nephrology,The Icahn School of Medicine at Mount Sinai

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Please note that some of the translations on this page are AI generated. Click here for the English version.

Overview

This article describes a method for isolating conditionally immortalized glomerular endothelial cells (GECs) from transgenic mice, allowing for the examination of mitochondrial structures in response to various stimuli. Utilizing high yields of GECs, the method serves as a foundation for screening potential therapies for diabetic kidney disease.

Key Study Components

Research Area

  • Cell biology
  • Diabetic kidney disease research
  • Endothelial cell biology

Background

  • Immortalized GECs provide a model for studying kidney functions.
  • The use of fluorescent mitochondria aids in observing intracellular changes.
  • Understanding GEC responses to stimuli can inform therapeutic strategies.

Methods Used

  • Isolation and culturing procedures for glomerular endothelial cells
  • Transgenic mice expressing fluorescent markers
  • Use of collagen IV and magnetic beads for cell selection

Main Results

  • A successful isolation protocol yielding viable GECs.
  • Verification of cell identity through CD31 positivity and mitochondrial fluorescence.
  • Demonstration of the method's application to drug screening in kidney disease.

Conclusions

  • The study illustrates a straightforward method for obtaining GECs from mice.
  • Results are relevant for understanding endothelial function and developing treatments for kidney diseases.

Frequently Asked Questions

What is the significance of using immortalized GECs?
They provide a stable model to study endothelial functions and responses.
How does the method help in diabetic kidney disease research?
It allows for screening potential therapies directly on GECs affected by the disease.
What precautions should be taken during the procedure?
Sterilization of equipment and workspace is crucial to prevent contamination.
Can the cells be preserved for future experiments?
Yes, the cells can be cryopreserved for later use.
What are CD31 positive cells?
They are a marker for endothelial cells, indicating successful isolation of GECs.
What role do the fluorescent mitochondria play in the study?
They allow researchers to visualize and evaluate mitochondrial changes in cells.

L’article décrit la méthode d’isolement des cellules endothéliales glomérulaires immortalisées conditionnellement des reins de souris transgéniques exprimant le virus simien thermolabile 40 et les mitochondries photo-activables, PhAMexcisée. Nous décrivons la procédure d’isolement des glomérules à partir de reins entiers à l’aide de billes, d’étapes de digestion, d’ensemencement et de culture de GECs-CD31 positifs.

Les cellules endothéliales glomérulaires immortalisées avec des mitochondries fluorescentes stables sont un excellent outil pour examiner l’effet de différents stimuli sur la structure mitochondriale in vitro. La méthode décrite dans le présent article produit un nombre élevé de cellules endothéliales glomérulaires. En testant de petites molécules sur un GEC, nous pourrions dépister en monothérapie pour la maladie rénale diabétique où les GEC sont affectés.

La méthode décrite est facile à réaliser et ne nécessite pas d’équipement spécifique. Cependant, il est important de suivre les étapes décrites et de stériliser l’équipement pour éviter toute contamination. Pour commencer, placez les matériaux de perfusion et d’isolement nécessaires pour isoler les cellules glomérulaires rénales sur un espace de travail.

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