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Biology
Aislamiento de células endoteliales glomerulares de ratón condicionalmente inmortalizadas con mit...
Aislamiento de células endoteliales glomerulares de ratón condicionalmente inmortalizadas con mit...
JoVE Journal
Biology
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JoVE Journal Biology
Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Aislamiento de células endoteliales glomerulares de ratón condicionalmente inmortalizadas con mitocondrias fluorescentes

Full Text
2,664 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.

El artículo describe el método para aislar células endoteliales glomerulares inmortalizadas condicionalmente de los riñones de ratones transgénicos que expresan el virus simio termolábil 40 y las mitocondrias fotoactivables, PhAMextirpado. Describimos el procedimiento para el aislamiento de glomérulos de riñones enteros utilizando perlas, pasos de digestión, siembra y cultivo de GECs-CD31 positivos.

Las células endoteliales glomerulares inmortalizadas con mitocondrias fluorescentes estables son una gran herramienta para examinar el efecto de diferentes estímulos sobre la estructura mitocondrial in vitro. El método descrito en el presente artículo produce un alto número de células endoteliales glomerulares. Al probar moléculas pequeñas en un GEC, podríamos detectar la monoterapia para la enfermedad renal diabética donde los GEC se ven afectados.

El método descrito es fácil de realizar y no requiere equipo específico. Sin embargo, es importante seguir los pasos descritos y esterilizar el equipo para evitar la contaminación. Para comenzar, coloque los materiales de perfusión y aislamiento necesarios para aislar las células glomerulares renales en un espacio de trabajo.

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