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

Medición de la masa mitocondrial y el potencial de la membrana en células madre hematopoyéticas y...

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

Medición de la masa mitocondrial y el potencial de la membrana en células madre hematopoyéticas y células T por citometría de flujo

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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.

Aquí describimos un método confiable para medir la masa mitocondrial y el potencial de membrana en células madre hematopoyéticas cultivadas ex vivo y células T.

Este protocolo es un ensayo basado en citometría de flujo simple que permite la medición del potencial de membrana mitocondrial y la masa mitocondrial en células vivas. Los ensayos metabólicos estándar fallan cuando se trabaja con poblaciones raras, como las células madre hematopoyéticas. Este método permite a los usuarios perfiles de membrana mitocondrial cuando se trabaja con un número bajo de células y para determinar nuevos moduladores metabólicos de células como los HSC.

Además, puede combinarlo con ensayos funcionales posteriores, como el trasplante de médula ósea o los ensayos formadores de colonias para determinar la funcionalidad de las células después del cultivo. Nuestra técnica puede ser explotada para la identificación de nuevas moléculas capaces de mejorar la función HSC en el contexto del trasplante de médula ósea y la recuperación inmune hematopoyética después de terapias ablativas. Como se describe en nuestro protocolo, nuestro método se puede utilizar para evaluar la aptitud metabólica de las células T inmunes.

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