Mitochondria are a distinctive, membrane-bound organelle present within all eukaryotic cells. As the primary site of ATP production and cellular energy supply, the unique aspects of mitochondria (including separate genetic material termed mDNA, it's own protein synthesizing machinery, and a strictly-regulated membrane potential charge gradient) make it an important subject of study. Additionally, as more aspects of the aging process and other human diseases are being discovered, the role of mitochondria has been shown to be even more prominent than previously suspected.
AAT Bioquest offers a wide range of imaging technologies for investigating mitochondrial morphology and functionality in live and fixed cells. The selective labeling of live cell compartments provides a powerful method for examining cellular events in a spatial and temporal context.
MitoLite™ Dyes for Live Cell Mitochondrial Imaging
MitoLite™ dyes are a series of cell-permeable cationic dyes used to label mitochondria in live cells. At micromolar concentrations (μM), MitoLite™ dyes passively diffuse across live cell membranes and selectively accumulate in active mitochondria via the mitochondrial membrane potential (ΔΨm) gradient. Since staining is lost during cell death due to the collapse (depolarization) of the mitochondrial membrane potential, MitoLite™ dyes can also be used to monitor cell viability. MitoLite™ dyes are available in spectral characteristics ranging from blue to near-infrared to accommodate multiplexing with other fluorescent probes or after fixation for co-localization studies. Retention after fixation varies amongst MitoLite™ dyes, with dyes such as MitoLite™ Blue FX490 being well-retained and MitoLite™ Green FM being lost after fixation. MitoLite™ dyes are key components of Cell Navigator™ Mitochondrion Staining Kits made available separately here. Each kit provides sufficient materials for 500 tests.
Features of MitoLite™ probes:
- Selectively accumulates in active mitochondria via the ΔΨm gradient
- Exceptionally bright fluorescence with high signal-to-noise ratios
- Photostable signal generation for extended imaging windows
- Can be imaged without washing
- Multiple wavelength options for multiplex analysis with other fluorescent probes
- Suitable for both proliferating and non-proliferating cells, as well as, cell in suspension and adherent cells
MitoLite™ Blue FX490
(Cat No. 22674)
MitoLite™ Green FM
(Cat No. 22695)
MitoLite™ NIR FX690
(Cat No. 22690)
Table 1. Available MitoLite™ dyes for labeling mitochondria in live cells.
|MitoLite™ Blue FX490||Live Cells||Yes||344||469||DAPI||500 Tests||22674||22665|
|MitoLite™ Green FM||Live Cells||No||508||528||FITC||10x50 µg||22695||-|
|MitoLite™ Green EX488||Live Cells||No||508||528||FITC||500 Tests||22675||22666|
|MitoLite™ Orange EX405||Live Cells||No||425||522||FITC||500 Tests||22679||22673|
|MitoLite™ Orange FX570||Live Cells||Yes||553||576||TRITC||500 Tests||22676||22667|
|MitoLite™ Red FX600||Live Cells||Yes||580||598||TRITC||500 Tests||22677||-|
|MitoLite™ Red CMXRos||Live Cells||Yes||579||599||TRITC||10x50 µg||22698||22668|
|MitoLite™ Deep Red FX660||Live Cells||Yes||640||659||Cy5||500 Tests||22678||22669|
|MitoLite™ NIR FX690||Live Cells||Yes||658||691||Cy5||500 Tests||22690||22670|
CytoFix™ Red Fixable Mitochondrial Stain
The fluorescence images of HeLa cells stained with CytoFix™ MitoRed in a 96-well black-wall clear-bottom plate. Image was acquired before (Left) and after (Right) fixation with 4% formaldehyde solution for 20 minutes at RT. The cells were imaged using fluorescence microscope with a Cy3/TRITC filter.
The enhanced cellular retention of CytoFix™ Red mitochondrial stain preserves its mitochondrial localized fluorescence even after the mitochondrial membrane potential is lost during fixation. The fluorescence signal generated by this dye is well-retained in the lyososme making it convenient for long-term mitochondrial tracking. CytoFix™ Red mitochondrial stain can be used with other fluorescent conjugates and other organelle stains for multicolor analysis. It can be used for both suspension and adherent cells and readily adapted for a wide variety of fluorescence platforms.
Features of CytoFix™ Red mitochondrial stain:
- High mitochondrial staining efficiency
- Long retention after fixation
- Labeling protocol with minimal hands on time
Table 2. CytoFix™ Red fixable mitochondrial stain
|CytoFix™ Red Mitochondrial Stain||Live Cells||Yes||540||590||Cy3/TRITC||500 Tests||23200|
Mitochondrial Membrane Potential Probes
The mitochondrial membrane potential (ΔΨm), generated by the electron transport chain, is a key parameter necessary for healthy mitochondrial functioning. Together with the proton gradient, it generates the driving force behind mitochondrial ATP synthesis. It plays a key role in mitochondrial homeostasis through selective elimination of dysfunctional mitochondria, and is an essential component of mitochondrial calcium homeostasis.
A distinctive feature of the early stages of apoptosis is the disruption of normal mitochondrial function. A collapse in mitochondrial membrane and redox potential may induce unwanted loss of cell viability and be a cause of various pathologies. We offer a wide assortment of fluorescent probes for analyzing aspects of normal mitochondrial activity in live cells, including reactive oxygen species (ROS) production, mitochondrial membrane potential and calcium flux.
JC-10™ Dual-Emission ΔΨm Probe
Camptothecin induced mitochondrial membrane potential changes were measured with JC-10™ (Cat No. 22204) and JC-1 (Cat No. 22200) in Jurkat cells. After Jurkat cells were treated with camptothecin (10 μM) for 4 hours, JC-1 and JC-10™ dye loading solutions were added to the wells and incubated for 30 minutes. The fluorescent intensities for both J-aggregates and monomeric forms of JC-1 and JC-10™ were measured at Ex/Em = 490/525 nm and 540/590 nm with NOVOstar microplate reader (BMG Labtech).
JC-10™, a derivative of JC-1, is potential-dependent probe used to determine ΔΨm by flow cytometry, fluorescence microscopy and in microplate-based fluorescent assays. In healthy cells, JC-10™ selectively accumulates in mitochondria generating orange J-aggregates that exhibit a broad excitation spectrum and emission maximum at 590 nm. However, in apoptotic and necrotic cells with low ΔΨm, JC-10™ diffuses out of the mitochondria and JC-10™ monomers are generated, resulting in a shift to green emission (525 nm). JC-10™ allows both qualitative and quantitative visualization of ΔΨm changes, considering the shift from orange to green fluorescence emission and the fluorescence intensity ratio, respectively.
Features of JC-10™:
- Easy-to-Use: JC-10™ does not percipitate when diluted into aqueous buffers, eliminating artifacts.
- Robust: JC-10™ has smaller assay deviations due to its enhanced solubility in aqueous media and higher sensitivity.
- Enhanced Signal: JC-10™ has a higher signal-to-background ratio than JC-1.
- Enhanced Sensitivity: JC-10™ has the ability to detect subtle changes in ΔΨm loss better than JC-1 in all tested cell lines.
- Broad Applications: JC-10™ can be used for primary rat hepatocytes.
- Convenient: JC-10™ is compatible with fluorescence microplate readers, cell imagers and flow cytometers.
Table 3. JC-10™ and JC-1 dual-emission mitochondrial membrane potential probes.
|JC-10™ *Superior alternative to JC-1*||508/524|
Table 4. Cell Meter™ JC-10™ assay kits for measuring mitochondrial membrane potential.
|Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Microplate Assays*||Microplate Reader||490/540 nm||525/590 nm||515/570 nm||500 tests||22800|
|Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Flow Cytometry Assays*||Flow Cytometer||488 nm laser||530/30|
Mitochondrial-Selective Rhodamine Esters
Cell-permeable cationic rhodamines, such TMRE and TMRM, are readily sequestered by active mitochondria, and commonly used to label mitochondria in living cells. Like JC-10™, TMRE and TMRM uptake in mitochondria is driven by the mitochondrial membrane potential. Both dyes have been successfully for dymanic and in situ
quantitative measurements, to screen for inhibitors of the mitochondrial transition pore, to assess the functionality of mitochondria in living cells, and can be used to discrimate between viable and non-viable cell populations. These potentiometric dyes exhibit minimal self-quenching, low cytotoxicity and have reasonable photostability, and their fluorescence intensities can be measured with either a flow cytometer or fluorescence microscope. In comparison to TMRM, TMRE is slightly more hydrophobic.
Table 5. Potentiometric dyes for measuring mitochondrial membrane potential.
|TMRE [Tetramethylrhodamine ethyl ester] *CAS#: 115532-52-0*||552||574||TRITC||25 mg||22220|
|TMRM [Tetramethylrhodamine methyl ester] *CAS#: 115532-50-8*||552||574||TRITC||25 mg||22221|