Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

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

doi:10.3791/64147

Isolamento de células endoteliais glomerulares de camundongos imortalizadas condicionalmente com ...

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Isolation of Conditionally Immortalized Mouse Glomerular Endothelial Cells with Fluorescent Mitochondria

Isolamento de células endoteliais glomerulares de camundongos imortalizadas condicionalmente com mitocôndrias fluorescentes

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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.

O artigo descreve o método de isolamento de células endoteliais glomerulares imortalizadas condicionalmente dos rins de camundongos transgênicos expressando o vírus símio termolábil 40 e mitocôndrias fotoativáveis,^{excisadas por PhAM}. Descrevemos o procedimento para isolamento de glomérulos de rins inteiros usando contas, etapas de digestão, semeadura e cultura de GECs-CD31 positivos.

Células endoteliais glomerulares imortalizadas com mitocôndrias fluorescentes estáveis são uma ótima ferramenta para examinar o efeito de diferentes estímulos na estrutura mitocondrial in vitro. O método descrito no presente artigo produz um elevado número de células endoteliais glomerulares. Ao testar pequenas moléculas em um GEC, poderíamos rastrear a monoterapia para a doença renal diabética, onde os GECs são afetados.

O método descrito é fácil de executar e não requer equipamento específico. No entanto, é importante seguir os passos descritos e esterilizar o equipamento para evitar a contaminação. Para começar, coloque os materiais de perfusão e isolamento necessários para isolar as células glomerulares renais em um espaço de trabalho.

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