Mitochondria are involved in various cellular processes, including autophagy, calcium homeostasis, immune response, signal transduction, and apoptosis. Mitochondrial homeostasis plays a critical role in regulating cellular morphology, quantity, subcellular distribution, and function.
During energy production, mitochondria store electrochemical potential across the inner mitochondrial membrane. The asymmetric distribution of protons and other ions across the membrane generates a mitochondrial membrane potential (MMP). Because of its electrical nature, this potential allows mitochondria in live cells to selectively retain specific dyes. Thus, membrane potential-dependent fluorescent dyes are commonly used to label mitochondria in live-cell imaging.
So, how do we choose between different dyes and probes? And how do we deal with abnormal labeling results? Based on practical experience, the factors that most affect labeling outcomes include:Cell type, Choice of fluorescent probe, and Experimental technique.
Let’s examine each aspect in detail.
Q1: Are mitochondrial morphologies the same across different cell types?
Below (Figure 1) shows confocal microscopy images of mitochondria in various cell types labeled with 30 nM TMRM (Tetramethylrhodamine methyl ester):
A. HMEC-1 (Human Microvascular Endothelial Cells): Mitochondria appear oval-shaped and highly branched, indicating high metabolic activity.
B. Mouse Astrocytes: Mitochondria are abundant and evenly distributed, reflecting high energy demand.
C. INS-1 Pancreatic β-cells: Mitochondria are densely packed, consistent with the energy needs during insulin secretion.
D. Freshly Isolated Rat Hepatocytes: Mitochondria are more diffusely distributed, which may relate to the complex metabolic functions of hepatocytes.
Figure 1. Confocal images of mitochondria in four different cell types labeled with 30 nM TMRM.
In Figure 2, MitoTracker Deep Red was used to label mitochondria in HepG2 (top), HeLa (middle), and COS-7 (bottom) cells. Morphological differences are again evident across cell types.
Figure 2. Confocal images of mitochondria in three cell types labeled with 100 nM MitoTracker Deep Red.
Q2: What are the types of mitochondrial dyes and how are they classified?
Common mitochondrial probes include:
MitoTracker series
MitoFluor series
JC-1 / JC-9 / JC-10
Rhodamine 123
TMRM
They can be broadly classified by their sensitivity to mitochondrial membrane potential (MMP):
1. MMP-Insensitive Probes:
MitoTracker Green FM: This dye reacts with thiol (-SH) groups on cysteine residues of mitochondrial proteins, enabling covalent labeling. It does not rely on membrane potential, making it suitable for studies of mitochondrial morphology (e.g., fusion and fission) and quantity. However, this dye cannot be fixed after staining as signal loss occurs.
MitoTracker Deep Red FM: Also MMP-insensitive, but can be fixed with 4% PFA and permeabilized with cold acetone after staining.
2. MMP-Sensitive Probes:
Rhodamine 123: Positively charged and accumulates in negatively charged, active mitochondria. Commonly used in apoptosis studies because mitochondrial depolarization during apoptosis leads to a significant drop in fluorescence. Cannot be fixed after staining.
MitoTracker Red CMXRos: Cell-permeant and selectively accumulates in active mitochondria, emitting fluorescence only in respiring cells. It reflects mitochondrial redox status and is suitable for assessing metabolic activity and oxidative stress. This probe can be fixed and permeabilized after staining.
Q3: Can mitochondrial labeling be done after fixation?
No, live-cell mitochondrial probes such as MitoTracker and TMRM are not suitable for fixed cells or tissue samples.
For fixed samples, it is recommended to use mitochondrial-specific antibodies, such as TOM20 or Grp75, in immunofluorescence assays.
Q4: Does working concentration affect staining results?
Absolutely. For different cell types and conditions, the dye concentration and incubation time significantly affect labeling efficiency. Common issues when using low concentrations include:
Weak or absent fluorescence
Low proportion of successfully labeled cells
Increasing the concentration of mitochondrial dyes often improves the labeling outcome significantly.
Q5: Can stained live cells be cultured further?
In general, yes—but only for a few hours.
Due to the nature of fluorescent probes, signal intensity will decay over time. One common observation is the loss of clear mitochondrial structure, often resulting in diffuse, punctate signals.
Q6: Can live-cell mitochondrial dyes be used on paraffin or frozen sections?
Paraffin sections: The fixation and embedding processes destroy membrane potential. Thus, live-cell dyes cannot be used. Instead, mitochondrial antibodies are recommended for immunodetection.
Frozen sections: In some cases, MitoTracker Deep Red can be attempted, depending on sample quality.
Q7: Can isolated mitochondria be labeled with live-cell dyes?
Yes, freshly isolated mitochondria with intact membrane potential can be labeled using live-cell probes such as:
MitoTracker series
Rhodamine 123
JC-1 / JC-9 / JC-10
However, frozen or previously stored mitochondria are not suitable, as their membrane integrity is often compromised.
References
Long Dan, Zhou Yanni, Feng Li, et al. Comparison of two mitochondrial labeling methods in cells and tissues. Journal of Sichuan University (Medical Science Edition), 2020, 51(3): 388–392. DOI: 10.12182/20200560205
Cottet-Rousselle C, Ronot X, Leverve X, Mayol JF. Cytometric assessment of mitochondria using fluorescent probes. Cytometry A. 2011 Jun;79(6):405–25. doi: 10.1002/cyto.a.21061. PMID: 21595013
Product Overview
|
Product Name |
Specification |
Cat No. |
|
1 mg/5 mg |
40705ES03/08 |
|
|
1 mg/5 mg |
40707ES03/08 |
|
|
JC-1 Mitochondrial Membrane Potential Assay Kit |
100 T |
40706ES60 |
|
Rhodamine 123 |
5 mg/25 mg |
40712ES08/25 |