High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

Ryan Awadhpersad; Christopher  B. Jackson

doi:10.3791/63000

Respirometria de alta resolução para avaliar bioenergésicos em células e tecidos usando respirôme...

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High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

Respirometria de alta resolução para avaliar bioenergésicos em células e tecidos usando respirômetros baseados em câmara e placas

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09:53 min

October 26, 2021

DOI: 10.3791/63000-v

Ryan Awadhpersad¹, Christopher B. Jackson¹

¹Department of Biochemistry and Developmental Biology, Faculty of Medicine,University of Helsinki

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Overview

This study investigates oxidative phosphorylation in mitochondria as a critical aspect of cellular energy metabolism. Using high-resolution respirometers, the research presents protocols for analyzing cellular energy metabolism and highlights the benefits of different respirometry devices.

Key Study Components

Research Area

Cellular energy metabolism
Oxidative phosphorylation
Mitochondrial dysfunction and disease

Background

Mitochondria are essential for energy production in cells.
Dysfunction in mitochondria is linked to various diseases.
High-resolution respirometry enables real-time insight into mitochondrial bioenergetics.

Methods Used

High-resolution respirometers for measuring oxygen consumption.
HEK293 cells as the primary biological model.
Protocols for both chamber and microplate-based assays.

Main Results

Establishment of protocols for consistent assessment of mitochondrial function.
Characterization of oxygen consumption in CRISPR-modified HEK293 cells.
Comparison of respiration rates between different tissue types.

Conclusions

The study validates the efficacy of high-resolution respirometry in assessing mitochondrial function.
Results contribute to understanding mitochondrial roles in various diseases.

Frequently Asked Questions

What is oxidative phosphorylation?

Oxidative phosphorylation is the metabolic pathway through which cells generate ATP, using oxygen to help convert nutrients into energy.

Why is mitochondrial function important?

Mitochondrial function is crucial for energy production and overall cellular metabolism, and dysfunctions can lead to various diseases.

How does high-resolution respirometry work?

High-resolution respirometry measures the oxygen consumption of cells to assess mitochondrial energy production in real-time.

What cell line was used in this study?

HEK293 cells were used as the biological model in this research.

What were the main findings related to CRISPR-knockouts?

The study found impaired respiration in CRISPR-knockout cell lines, indicating deficiencies in oxidative phosphorylation.

What is the significance of comparing tissue types?

Comparing tissues, such as mouse cerebellum and soleus muscle, provides insights into the varying capacities for mitochondrial respiration and energy production across different cell types.

How do the methods used contribute to biological research?

These methods enhance our understanding of cellular metabolism, helping to diagnose and explore therapies for mitochondrial diseases.

Avaliar a fosforilação oxidativa usando respirômetros de alta resolução tornou-se parte integrante da análise funcional das mitocôndrias e do metabolismo de energia celular. Aqui, apresentamos protocolos para a análise do metabolismo de energia celular usando respirômetros de alta resolução baseados em câmara e microplatos e discutimos os principais benefícios de cada dispositivo.

Mitocôndrias fornecem energia celular através da fosforilação oxidativa contribuindo para quase todos os processos dentro da célula. Consequentemente, as disfunções mitocondriais estão associadas a um amplo espectro de doenças. A principal vantagem dessa técnica é a medição em tempo real dos bioenergésicos mitocondriais pelo consumo de oxigênio e, portanto, uma avaliação precisa do metabolismo total da energia celular.

A respirometria de alta resolução permite uma avaliação direta do que no sistema de fosforilação oxidativa está falhando, potencialmente fornecendo pistas diagnósticas em doenças mitocondriais primárias e disfunções mitocondriais secundárias associadas a muitos distúrbios. Demonstrando o procedimento será Ryan Awadhpersad, doutorando no meu laboratório. Para começar, realize a calibração de oxigênio executando os respirômetros em 2,1 mililitros de meio de respiração mitocondrial a 37 graus Celsius por mais de 45 minutos e proceda se a variação da linha de base estiver dentro de quatro picomoles por segundo.

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