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用荧光线粒体分离条件永生化的小鼠肾小球内皮细胞
用荧光线粒体分离条件永生化的小鼠肾小球内皮细胞
JoVE Journal
Biology
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JoVE Journal Biology
Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

用荧光线粒体分离条件永生化的小鼠肾小球内皮细胞

Full Text
2,807 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.

本文描述了从表达热不稳定猿病毒40和光激活线粒体PhAM切除的转基因小鼠的肾脏中分离条件永生化肾小球内皮细胞的方法。我们描述了使用磁珠、消化步骤、播种和培养 GEC-CD31 阳性从整个肾脏中分离肾小球的程序。

具有稳定荧光线粒体的永生化肾小球内皮细胞是体外检查不同刺激对线粒体结构影响的好工具。本文中描述的方法产生大量的肾小球内皮细胞。通过在GEC上测试小分子,我们可以筛查GEC受到影响的糖尿病肾病的单一疗法。

所描述的方法易于执行,不需要特定的设备。但是,重要的是要遵循所述步骤并对设备进行消毒以避免污染。首先,将分离肾小球细胞所需的灌注和分离材料放置在工作空间上。

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