Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis
Abstract
1. Introduction
2. Results
2.1. Development of a Biological Assay for the Quantitative Assessment of Mitophagy in Individual Mitochondria
2.2. Flow Cytometry-Based Assessment of Mitochondria-Containing Extracellular Vesicles in THP1 Macrophages
2.3. Flow Cytometry-Based Analysis of Intact, Cell-Free Mitochondria from Blood
2.4. Result Summary
3. Discussion
4. Materials and Methods
4.1. Animals and Cell Lines
4.2. Sample Generation
4.2.1. From Blood (Cell-Free Mitochondria)
4.2.2. From Plated Cells
4.2.3. From Cell Culture Media
4.3. Flow Cytometric Analysis and Sorting
4.4. Downstream Applications
4.4.1. Single-Molecule PCR (smPCR)
4.4.2. ATP Assay
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
FAMS | Fluorescence-activated mitochondria sorting |
ΔΨM | Mitochondrial membrane potential |
mtDNA | Mitochondrial DNA |
ROS | Reactive oxygen species |
EV | Extracellular vesicle |
PolG | Polymerase gamma |
MTG | MitoTracker Green FM |
PINK1 | Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 |
smPCR | Single-molecule PCR |
SEM | Standard error of the mean |
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Experimental Aim | Experimental Approach | Result |
---|---|---|
Development of a biological assay for the quantitative assessment of mitophagy in individual mitochondria without the use of membrane uncouplers. | Pol G mutant fibroblast mitochondria were assessed for ΔΨM, PINK1, and Parkin expression using FAMS. Individual mitochondria were sorted and assessed for mtDNA copy number. | PINK1 and Parkin expression was detected on a per organelle basis without the use of membrane uncouplers and were shown to colocalize at different rates based on mitochondrial subpopulation. |
Assessment of spent cell culture media to determine whether detected cell-free mitochondria were free-floating or contained within extracellular vesicles. | Following a 24 h incubation of THP-1 macrophages, spent media were collected, ensured to be free of cells, and assessed for the presence of free mitochondria and extracellular vesicles using FAMS. | Both free-floating and vesicle-enclosed mitochondria were detected. The detection of vesicle-enclosed mitochondria raises the question of whether these organelles are being intentionally excreted by the macrophages in vitro. |
Test for the presence of intact, cell-free mitochondria in mouse blood plasma. | Plasma was collected and tested for the presence of respiratory-competent mitochondria using FAMS. Sorted mitochondria were pooled and tested for the ability to produce ATP. | Cell-free, respiratory-competent mitochondria were detected in mouse plasma using FAMS. Their ability to respire was confirmed by ATP assay. |
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Piasecki, A.J.; Sheehan, H.C.; Tilly, J.L.; Woods, D.C. Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis. Int. J. Mol. Sci. 2025, 26, 5481. https://doi.org/10.3390/ijms26125481
Piasecki AJ, Sheehan HC, Tilly JL, Woods DC. Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis. International Journal of Molecular Sciences. 2025; 26(12):5481. https://doi.org/10.3390/ijms26125481
Chicago/Turabian StylePiasecki, Andrew J., Hannah C. Sheehan, Jonathan L. Tilly, and Dori C. Woods. 2025. "Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis" International Journal of Molecular Sciences 26, no. 12: 5481. https://doi.org/10.3390/ijms26125481
APA StylePiasecki, A. J., Sheehan, H. C., Tilly, J. L., & Woods, D. C. (2025). Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis. International Journal of Molecular Sciences, 26(12), 5481. https://doi.org/10.3390/ijms26125481