Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Rihab Bouchareb; Liping Yu; Emelie Lassen; Ilse S. Daehn

doi:10.3791/64147

JoVE Journal
Biology

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Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

蛍光ミトコンドリアによる条件付き不死化マウス糸球体内皮細胞の単離

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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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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.

この記事では、熱不安定性サルウイルス40および光活性化可能なミトコンドリア、PhAM^{を切除した}トランスジェニックマウスの腎臓から条件付き不死化糸球体内皮細胞を単離する方法について説明しています。ビーズを用いた腎臓全体からの糸球体単離の手順、消化ステップ、播種、およびGECs-CD31陽性の培養について説明します。

安定した蛍光ミトコンドリアを有する不死化糸球体内皮細胞は、in vitroでミトコンドリア構造に対するさまざまな刺激の影響を調べるための優れたツールです。本論文に記載されている方法は、多数の糸球体内皮細胞を生じる。GECで低分子を試験することにより、GECが罹患している糖尿病性腎疾患の単剤療法をスクリーニングすることができます。

説明されている方法は実行が簡単で、特定の機器を必要としません。ただし、汚染を避けるために、説明されている手順に従い、機器を滅菌することが重要です。まず、腎糸球体細胞を単離するために必要な灌流および分離材料をワークスペースに配置します。

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