Mitochondria

Probes for Mitochondria

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.

CytoFix™ Red Fixable Mitochondrial Stain

Table 2. CytoFix™ Red fixable mitochondrial stain

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

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.

Figure 3. 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).

Table 3. JC-10™ and JC-1 dual-emission mitochondrial membrane potential probes.

Table 4. Cell Meter™ JC-10™ assay kits for measuring mitochondrial membrane potential.

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.

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