Mitochondrial Apoptosis Assays for Flow Cytometry
A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. Life Technologies™ exclusively offers a number of Life Technologies™ fluorescent probes for analyzing mitochondrial activity in live cells by flow cytometry, with minimal disruption of cellular function.
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The MitoProbe™ family of mitochondrial stains provide quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis.
- Easy to use and compatible with existing research protocols
- Can be used with multiple cell types or laser excitations
- Available for 488 nm and 633/635 nm excitation
- Low compensation alternatives
|Figure 1. Detection of changes in mitochondrial membrane potential using the MitoProbe™ DiIC1(5) Assay Kit. Decrease in mitochondrial membrane potential as demonstrated with DiIC1(5) fluorescence due to the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Jurkat cells (T-cell leukemia, human) were stained with 50 nM DiIC1(5) alone (blue) or in the presence of 50 μM CCCP (red).|
MitoTracker® dyes are membrane potential–dependent probes for staining mitochondria in live cells. The fluorescence signal of MitoTracker® dyes is brighter in active mitochondria than in mitochondria with depolarized membranes, providing a way to identify healthy cells in a population.
The staining pattern of MitoTracker® dyes is retained throughout subsequent flow cytometry immunocytochemistry, DNA end labeling, in situ hybridization, or counterstaining steps. MitoTracker® Red CMXRos exhibits minimal spectral overlap when paired with a green-fluorescent dye, while MitoTracker® Orange CMTMRos is compatible with far-red–fluorescent dyes.
Figure 2. Analysis of decrease in mitochondrial membrane potential during apoptosis using MitoTracker® Red dye. Jurkat cells (T-cell leukemia, human) were treated in complete medium with 10 μM camptothecin for four hours (A) or untreated (B). Annexin V-Alexa Fluor® 488 was added to detect changes in membrane asymmetry and MitoTracker® Red dye was added to detect changes in mitochondrial membrane potential. Note that apoptotic cells show higher reactivity for annexin V and lower MitoTracker® Red dye fluorescence than do live cells.
The mitochondrial permeability transition pore (MPTP) is a nonspecific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death. This assay provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone.
Figure 3. Flow cytometric analysis of MPTP using the MitoProbe™ Transition Pore Assay Kit. Jurkat cells were incubated with the reagents in the MitoProbe™ Transition Pore Assay Kit and analyzed by flow cytometry. In the absence of CoCl2 and ionomycin, fluorescent calcein is present in the cytosol as well as the mitochondria, resulting in a bright signal (left panel). In the presence of CoCl2, calcein in the mitochondria emits a signal, but the cytosolic calcein fluorescence is quenched; the overall fluorescence is reduced compared to calcein alone (center panel). When ionomycin (a calcium ionophore) and CoCl2 are added to the cells at the same time as calcein AM, the fluorescence signals from both the cytosol and mitochondria are largely abolished (right panel).
More Flow Cytometry Assays for Apoptosis
For Research Use Only. Not for use in diagnostic procedures.