Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11120%2F21%3A43921946" target="_blank" >RIV/00216208:11120/21:43921946 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/00216208:11150/21:10430541

Výsledek na webu

<a href="https://doi.org/10.3791/62902" target="_blank" >https://doi.org/10.3791/62902</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.3791/62902" target="_blank" >10.3791/62902</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Popis výsledku v původním jazyce

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.

Název v anglickém jazyce

Measuring Mitochondrial Substrate Flux in Recombinant Perfringolysin O-Permeabilized Cells

Popis výsledku anglicky

Mitochondrial substrate flux is a distinguishing characteristic of each cell type, and changes in its components such as transporters, channels, or enzymes are involved in the pathogenesis of several diseases. Mitochondrial substrate flux can be studied using intact cells, permeabilized cells, or isolated mitochondria. Investigating intact cells encounters several problems due to simultaneous oxidation of different substrates. Besides, several cell types contain internal stores of different substrates that complicate results interpretation. Methods such as mitochondrial isolation or using permeabilizing agents are not easily reproducible. Isolating pure mitochondria with intact membranes in sufficient amounts from small samples is problematic. Using non-selective permeabilizers causes various degrees of unavoidable mitochondrial membrane damage. Recombinant perfringolysin O (rPFO) was offered as a more appropriate permeabilizer, thanks to its ability to selectively permeabilize plasma membrane without affecting mitochondrial integrity. When used in combination with microplate respirometry, it allows testing the flux of several mitochondrial substrates with enough replicates within one experiment while using a minimal number of cells. In this work, the protocol describes a method to compare mitochondrial substrate flux of two different cellular phenotypes or genotypes and can be customized to test various mitochondrial substrates or inhibitors.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

30105 - Physiology (including cytology)

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

JoVE - Journal of Visualized Experiments

ISSN

1940-087X

e-ISSN

—

Svazek periodika

174

Číslo periodika v rámci svazku

August

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

16

Strana od-do

"e62902"

Kód UT WoS článku

000709869500003

EID výsledku v databázi Scopus

2-s2.0-85114358485