Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Moustafa Elkalaf; Karol&#237;na Van&#283;&#269;kov&#225;; Pavla Sta&#328;kov&#225;; Zuzana &#268;ervinkov&#225;; Jan Pol&#225;k; Otto Ku&#269;era

doi:10.3791/62902

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

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Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

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06:17 min

August 13, 2021

DOI: 10.3791/62902-v

Moustafa Elkalaf^1,2, Karolína Vaněčková^1,2, Pavla Staňková¹, Zuzana Červinková¹, Jan Polák*^2,3, Otto Kučera*¹

¹Department of Physiology, Faculty of Medicine in Hradec Králové,Charles University, ²Department of Pathophysiology, Third Faculty of Medicine,Charles University, ³Department of Internal Medicine,University Hospital Kralovske Vinohrady

Overview

This study presents a modified protocol for assessing mitochondrial respiratory substrate flux using recombinant perfringolysin O in conjunction with microplate-based respirometry. The research highlights the impact of metformin on mitochondrial respiration across two distinct tumor cell lines, A549 and Hep G2.

Key Study Components

Research Area

Mitochondrial metabolism
Cellular response to pharmacological treatments
Oncology and cancer research

Background

The importance of mitochondrial function in various pathologies
How drugs like metformin influence cancer cell metabolism
Previous methods to assess mitochondrial respiration

Methods Used

Microplate-based respirometry
A549 and Hep G2 tumor cell lines
Recombinant perfringolysin O for permeabilization

Main Results

Metformin treatment increased succinate-induced respiration in A549 cells compared to Hep G2 cells.
Similar enhancements observed in pyruvate malate and glutamate malate induced respiration.
The findings validate the potential of utilizing mitochondrial respiration assays to explore drug impacts in cancer research.

Conclusions

The study demonstrates a novel method for evaluating drug effects on mitochondrial function in cancer cells.
This could facilitate further research into metabolic targeting in oncology.

Frequently Asked Questions

What are the advantages of using microplate respirometry?

Microplate respirometry allows for high-throughput screening, requiring minimal samples while providing reliable data on mitochondrial function.

How does metformin affect mitochondrial respiration?

Metformin alters mitochondrial respiration, which can reveal important insights into the metabolic adaptation of cancer cells.

What cell lines were used in this study?

A549 and Hep G2 tumor cell lines were analyzed for their mitochondrial respiration responses to metformin treatment.

Why is recombinant perfringolysin O important in this protocol?

Recombinant perfringolysin O is used to permeabilize cell membranes, facilitating the assessment of mitochondrial function without the interference of cellular metabolic processes.

What was a key finding regarding mitochondrial respiration between the two cell lines?

A549 cells exhibited a higher rate of mitochondrial respiration induced by succinate when compared to Hep G2 cells.

Can this method be applied to other types of cancer research?

Yes, this method is versatile and can be adapted to study various cancer types and other conditions affecting mitochondrial metabolism.

How is the assay prepared before measurement?

The assay involves cell seeding, treatment with substrates, and calibration of the respirometry analyzer to ensure accurate results.

In this work, we describe a modified protocol to test mitochondrial respiratory substrate flux using recombinant perfringolysin O in combination with microplate-based respirometry. With this protocol, we show how metformin affects mitochondrial respiration of two different tumor cell lines.

Because it tests mitochondria substrate flux that can be affected with different pathologies or treatments. For example, today we will use it to reveal cancer cell response to Metformin treatment. It requires a minimal number of cells while having sufficient replicates and appropriate control for each distinct material.

The analyzer is for research use only. This technique can identify errors in mitochondrial metabolism and can be used to evaluate warriors drugs affecting mitochondria. Demonstrating the procedure will be Karolina Vaneckova an undergraduate student and research technician from my laboratory.

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