Comparison of methods of mitochondrial content assessment in HSCs
| Probe/Technique . | Product description . | Use . | Pros . | Cons . |
|---|---|---|---|---|
| MitoTracker green | Green-fluorescent dye that covalently binds to mitochondrial proteins | Mitochondrial mass | (1) Easy to use (2) Applicable to flow cytometry (3) Can be used to assess mitochondrial morphology by IF in cells with positive MMP73 | (1) Binding to mitochondria depends on the MMP and mitochondrial OXPHOS activity25 ; hence, poor method to assess mitochondrial mass in cells with low MMP such as HSCs (2) Dye is excluded from cells with xenobiotic efflux pump activity15 (3) Does not work on fixed cells (4) Cannot be used to assess mitochondrial morphology in HSCs because it gives poor resolution by IF due to lack of retention of the dye onto mitochondria (Ashwini Hinge and M.-D.F., unpublished data) |
| Rhodamine 123 | Cell-permeant, cationic, green-fluorescent dye that is readily sequestered by active mitochondria | MMP | Dye is excluded from cells with xenobiotic efflux pump activity24 | |
| qPCR for mtDNA | mtDNA, proxy for mitochondrial mass | (1) Quantitative method (2) Independent of the MMP and mitochondrial OXPHOS activity | (1) Inaccurate and variable results when using a low number of cells (1000) (Ashwini Hinge and M.-D.F., unpublished data) (2) mtDNA quantification is not directly proportional to mitochondrial mass because mitochondria contain multiple DNA copies and because mtDNA loss is uncoupled to mitochondrial body23,74 | |
| Electron microscopy | Mitochondrial structure | (1) Information on cristae structure (2) Independent of the mitochondrial activity and membrane potential | 2D EM provides information on one single plan per cell, which is insufficient to capture the entire mitochondrial network in round cells like HSC26 | |
| Immunofluorescence | Immunostaining for mitochondrial protein like Tom20 | (1) Mitochondrial shape and morphology (2) Mitochondrial content | (1) Independent of mitochondrial activity and membrane potential (2) High-resolution confocal imaging with 3D reconstruction for information on the entire mitochondrial network (3) Assessment of mitochondrial mass with MFI measurement | Highly dependent on fixation/staining conditions; works well with 2% PFA and 0.05% Triton for 1-2 min15,26 ; classical limitations of image quantification of fluorescence signals |
| Probe/Technique . | Product description . | Use . | Pros . | Cons . |
|---|---|---|---|---|
| MitoTracker green | Green-fluorescent dye that covalently binds to mitochondrial proteins | Mitochondrial mass | (1) Easy to use (2) Applicable to flow cytometry (3) Can be used to assess mitochondrial morphology by IF in cells with positive MMP73 | (1) Binding to mitochondria depends on the MMP and mitochondrial OXPHOS activity25 ; hence, poor method to assess mitochondrial mass in cells with low MMP such as HSCs (2) Dye is excluded from cells with xenobiotic efflux pump activity15 (3) Does not work on fixed cells (4) Cannot be used to assess mitochondrial morphology in HSCs because it gives poor resolution by IF due to lack of retention of the dye onto mitochondria (Ashwini Hinge and M.-D.F., unpublished data) |
| Rhodamine 123 | Cell-permeant, cationic, green-fluorescent dye that is readily sequestered by active mitochondria | MMP | Dye is excluded from cells with xenobiotic efflux pump activity24 | |
| qPCR for mtDNA | mtDNA, proxy for mitochondrial mass | (1) Quantitative method (2) Independent of the MMP and mitochondrial OXPHOS activity | (1) Inaccurate and variable results when using a low number of cells (1000) (Ashwini Hinge and M.-D.F., unpublished data) (2) mtDNA quantification is not directly proportional to mitochondrial mass because mitochondria contain multiple DNA copies and because mtDNA loss is uncoupled to mitochondrial body23,74 | |
| Electron microscopy | Mitochondrial structure | (1) Information on cristae structure (2) Independent of the mitochondrial activity and membrane potential | 2D EM provides information on one single plan per cell, which is insufficient to capture the entire mitochondrial network in round cells like HSC26 | |
| Immunofluorescence | Immunostaining for mitochondrial protein like Tom20 | (1) Mitochondrial shape and morphology (2) Mitochondrial content | (1) Independent of mitochondrial activity and membrane potential (2) High-resolution confocal imaging with 3D reconstruction for information on the entire mitochondrial network (3) Assessment of mitochondrial mass with MFI measurement | Highly dependent on fixation/staining conditions; works well with 2% PFA and 0.05% Triton for 1-2 min15,26 ; classical limitations of image quantification of fluorescence signals |
Description of various methods that have been used to measure mitochondrial mass in HSCs. The pros and cons are mostly applicable to HSCs. These methods are, however, classically used successfully in other tissues and cell types to measure mitochondrial content.
3D, 3-dimensional; EM, electron microscopy; IF, immunofluorescence; MFI, mean fluorescence intensity; mtDNA, mitochondrial DNA; PFA, paraformaldehyde; qPCR, quantitative polymerase chain reaction.