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Biology
Şartlı Ölümsüzleştirilmiş Fare Glomerüler Endotel Hücrelerinin Floresan Mitokondri ile İzolasyonu
Şartlı Ölümsüzleştirilmiş Fare Glomerüler Endotel Hücrelerinin Floresan Mitokondri ile İzolasyonu
JoVE Journal
Biology
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JoVE Journal Biology
Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Şartlı Ölümsüzleştirilmiş Fare Glomerüler Endotel Hücrelerinin Floresan Mitokondri ile İzolasyonu

Full Text
2,787 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.

Makalede, termolabil simian virüsü 40'ı eksprese eden transgenik farelerin böbreklerinden şartlı olarak ölümsüzleştirilmiş glomerüler endotel hücrelerinin izole edilmesi ve foto-aktive edilebilir mitokondri, PhAMeksize edilmesi yöntemi açıklanmaktadır. Boncuklar, sindirim adımları, tohumlama ve GEC-CD31 pozitifin kültürlenmesi kullanılarak tüm böbreklerden glomerül izolasyonu prosedürünü açıklıyoruz.

Stabil floresan mitokondrili ölümsüzleştirilmiş glomerüler endotel hücreleri, farklı uyaranların in vitro mitokondriyal yapı üzerindeki etkisini incelemek için harika bir araçtır. Bu makalede açıklanan yöntem, çok sayıda glomerüler endotel hücresi vermektedir. Bir GEC üzerinde küçük molekülleri test ederek, GEC'lerin etkilendiği diyabetik böbrek hastalığı için monoterapi taraması yapabiliriz.

Açıklanan yöntemin gerçekleştirilmesi kolaydır ve belirli bir ekipman gerektirmez. Bununla birlikte, açıklanan adımları takip etmek ve kontaminasyonu önlemek için ekipmanı sterilize etmek önemlidir. Başlamak için, böbrek glomerüler hücrelerini izole etmek için gereken perfüzyon ve izolasyon malzemelerini bir çalışma alanına yerleştirin.

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