Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

Mukul Girotra; Anne-Christine Thierry; Alexandre Harari; George Coukos; Olaia Naveiras; Nicola Vannini

doi:10.3791/60475

Mesure de la masse mitochondriale et du potentiel membranaire dans les cellules souches hématopoï...

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Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

Mesure de la masse mitochondriale et du potentiel membranaire dans les cellules souches hématopoïétiques et les lymphocytes T par cytométrie de flux

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07:57 min

December 26, 2019

DOI: 10.3791/60475-v

Mukul Girotra*^1,2, Anne-Christine Thierry³, Alexandre Harari^1,3, George Coukos¹, Olaia Naveiras^2,4, Nicola Vannini*¹

¹Department of Oncology UNIL CHUV, Ludwig Institute for Cancer Research Lausanne,University of Lausanne, ²Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences,Swiss Federal Institute of Technology Lausanne (EPFL), ³Center of Experimental Therapeutics, Department of Oncology,Centre Hospitalier Universitaire Vaudois, ⁴Hematology Service, Department of Oncology,Centre Hospitalier Universitaire Vaudois (CHUV)

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Overview

This study presents a reliable flow cytometry-based assay for measuring mitochondrial mass and membrane potential in living ex vivo cultured hematopoietic stem cells (HSCs) and T cells. The method is particularly useful for analyzing rare cell populations and can be combined with functional assays to evaluate HSC functionality.

Key Study Components

Research Area

Cell Biology
Hematopoietic Stem Cells
Metabolic Profiling

Background

Standard metabolic assays are ineffective for rare cell populations.
Understanding mitochondrial function is crucial for enhancing HSC functionality.
Previous literature lacks detailed protocols for low cell numbers.

Methods Used

Flow cytometry for mitochondrial profiling
Ex vivo cultured hematopoietic stem cells and T cells
Use of nicotinamide riboside and TMRM staining for evaluating mitochondrial status

Main Results

Nicotinamide riboside treatment increased TMRM low population and decreased TMRM fluorescence intensity in HSCs.
Mitochondrial mass remained unchanged with treatment.
The method successfully identifies specific cell populations through FACS analysis.

Conclusions

This study provides a critical method for assessing metabolic fitness in HSCs.
It's relevant for improving HSC function in bone marrow transplants and other therapeutic contexts.

Frequently Asked Questions

What is the purpose of this protocol?

To measure mitochondrial mass and membrane potential in low numbers of hematopoietic stem cells and T cells.

How does the method benefit research on stem cells?

It enables the analysis of mitochondrial metabolism, which is vital for HSC functionality.

Can this method be used with other assays?

Yes, it can be combined with downstream assays like bone marrow transplantation and colony-forming assays.

What precautions should be taken during the process?

Minimize exposure to light after staining and be cautious with the use of methotrexate, as it can perturb ionic equilibrium.

What results can be observed using this protocol?

Increased mitochondrial membrane potential and evaluation of mitochondrial mass can be observed with the right treatments.

What technologies are key to this method?

Flow cytometry and fluorescent staining techniques are essential for the protocol.

Is visual demonstration of the method important?

Yes, visual demonstrations can help in understanding critical steps that are often omitted in traditional literature.

Ici nous décrivons une méthode fiable pour mesurer la masse mitochondriale et le potentiel de membrane dans les cellules souches hématopoïétiques et les cellules T cultivées ex vivo.

Ce protocole est un essai simple basé sur la cytométrie d’écoulement permettant la mesure du potentiel mitochondrique de membrane et de la masse mitochondrique dans les cellules vivantes. Les analyses métaboliques standard échouent lorsque l’on travaille avec des populations rares telles que les cellules souches hématopoïétiques. Cette méthode permet aux utilisateurs de profiler la membrane mitochondriale lorsqu’ils travaillent avec un faible nombre de cellules et de déterminer de nouveaux modulateurs métaboliques de cellules telles que les HSC.

En outre, vous pouvez le combiner avec des analyses fonctionnelles en aval telles que la transplantation de moelle osseuse ou des tests de formation de colonies pour déterminer la fonctionnalité de vos cellules post-culture. Notre technique peut être exploitée pour l’identification de nouvelles molécules capables d’améliorer la fonction HSC dans le contexte de la transplantation de moelle osseuse et de la récupération immunitaire hématopoïétique après des thérapies ablatives. Comme décrit dans notre protocole, notre méthode peut être employée pour évaluer la forme physique métabolique des cellules immunisées de T.

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