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A distinctive feature of apoptosis is the disruption of normal mitochondrial function, especially changes that affect the mitochondrial membrane potential (ΔΨm).
ΔΨm is critical for maintaining the physiological function of the respiratory chain to generate ATP; the opening the mitochondrial permeability transition pore (MPTP) leads to the collapse of the ΔΨm and subsequent release of cytochrome C into the cytosol.
Mitochondrial membrane potential is commonly detected using cationic (positively-charged) fluorescent dyes that accumulate in the negatively-charged mitochondrial matrix. The dye accumulates in inverse proportion to ΔΨm i.e. the more negative the ΔΨm, the more dye accumulates. This means that a healthy cell will contain more dye while an apoptotic cell will contain less. These dyes can be used qualitatively in fluorescence microscopy or quantitatively in flow cytometry or microplate spectrophotometry.
The table below highlights the mitochondrial membrane potential probes available from us:
|Probe||Healthy/ apoptotic cells||Ex/Em||Best to use for:|
|TMRE: Cationic red-orange dye that readily accumulated in active mitochondria. Depolarized or inactive mitochondria have decreased membrane potential and fail to sequester TMRE.|
Healthy cells: bright orange fluorescence
Apoptotic cells: weak orange fluorescence
Ex = 549 nm
Em = 575 nm
|Time-lapse fluorescence microscopy and immunofluroescence staining|
|JC-1: Cationic green-red dye that exhibits potential- dependent accumulation in mitochondria. Mitochondrial depolarization is indicated by a decrease in the red/ green fluorescence intensity ratio.|
Healthy cells: red
Apoptotic cells: green
Ex = 530 nm
Em = 590 nm
|Flow cytometry or microplate spectrophotometry. Ideal for comparative measurements.|
|JC-10: a derivative of JC-1, has improved solubility in aqueous media and the ability to detect subtler changes in mitochondrial membrane potential loss.|
Healthy cells: orange fluorescence.
Apoptotic cells: green
Ex = 490 nm
Em = 520 - 570 nm
|Flow cytometry or microplate spectrophotometry. Ideal for comparative measurements|
|MitoOrange dye and MitoNIR dye: cationic dyes. In normal cells, the fluorescence intensity increases when dye accumulates in normal cells. In apoptotic cells, the fluorescence intensity of the dye decreases following the collapse of the Δψm.|
Healthy cells: red or near infrared (NIR) fluorescence.
Apoptotic cells: weak.
MitoOrange: Ex = 540 nm
Em = 590 nm
MitoRed: Ex = 635 nm
Em = 660 nm
|Flow cytometry or micrplate spectrophotometry. Multiparametric study of apoptosis.|
|FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) (ab120081) is an ionophore uncoupler of oxidative phosphorylation. Treating cells with FCCP eliminates mitochondrial membrane potential, which makes FCCP a very good positive control for these type of studies.|
TMRE - Mitochondrial membrane potential assay kit (ab113852): HeLa (A) and Jurkat cells (B) were stained with 200 nm TMRE for 20 minutes and immediately imaged.
Cytochrome C release
The collapse of the ΔΨm is a fairly catastrophic event. It leads to the opening of the mitochondrial permeability transition pores in the mitochondrial membrane, and the subsequent release of cytochrome C in the cytosol, which in turn triggers other downstream events in the apoptotic cascade.
Once the mitochondrial pores are opened and cytochrome C is released, the apoptotic cascade reaches a “point of no return” from which it is very unlikely that the cell can recover and death is the most likely outcome.
The most common technique to detect cytochrome C release is through western blot on protein extracted from different subcellular compartments. It is very important in this case to ensure that the different subcellular fractions are not contaminated with other fractions. This can be easily checked with specific and reliable subcellular markers:
Apotrack™ Cytochrome C Apoptosis WB Antibody Cocktail (ab110415): This western blot antibody cocktail allows the detection of cytochrome C in cytoplasmic and mitochondrial fractions. The product contains a set of organelle control markers to help you monitor and optimize your fractionation protocol.
On the other hand, immunofluorescence staining of fixed cells with a cytochrome C antibody at selected time points can be used to visualize cytochrome C release from the mitochondria into the cytoplasm.
Apotrack™ Cytochrome C Apoptosis ICC Antibody Cocktail (ab110417): HeLa cells treated with staurosporine were stained with anti-cytochrome C antibody (green) and anti-ATP synthase subunit alpha antibody (red). The white arrow indicated cytochrome C release from mitochondria.