Mitochondrial Permeability Transition Detection Kits

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Detection of the mitochondrial permeability transition event (PT) provides an early indication of the initiation of cellular apoptosis.  This process is typically defined as a collapse in the electrochemical gradient across the mitochondrial membrane, as measured by the change in the membrane potential (DY).

Changes in the mitochondrial DY lead to the insertion of proapoptotic proteins into the membrane and possible oligomerisation of BID, BAK, BAX or BAD.  This could create pores, which dissipate the transmembrane potential, thereby releasing cytochrome c into the cytoplasm.

Loss of mitochondrial DY, indicative of apoptosis, can be detected by a unique fluorescent cationic dye,5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-
benzamidazolocarbocyanin iodide, commonly known as JC-1.  This dye has been incorporated into the user-friendly MitoPTTM kit for the simple and reproducible detection of the PT event in apoptotic cells.

The structure of the MitoPTTM reagent allows it to easily penetrate cells and healthy mitochondria.  Once inside a healthy non-apoptotic cell, the lipohillic MitoPTTM reagent, bearing a delocalised positive charge, enters the negatively charged mitochondria where it aggregates and fluoresces red.  These aggregates, first described by Jelley in 1937, are referred to as J-aggregates.  When the mitochondrial AY collapses in apoptotic cells, the MitoPTTM reagent no longer accumulates inside the mitochrondria.  Instead, it is distributed throughout the cell.  When dispersed in the manner, the MitoPTTM  reagent assumes a monomeric form, which fluoresces green.  Use of the MitoPTTM kit allows the easy distinction between non-apoptotic red fluorescent cells and apoptotic green fluorescent cells.

The MitoPTTM kit can evaluate apoptosis using three different technologies: flow cytometers, fluorometric plate readers, and fluorescence microscopes.

Fluorescence Microscopy

 

Jurkat cells were stained with MitoPTTM  and viewed through a fluorescence microscope using a broad band path filter.  Non-apoptotic cells exhibit red stained mitochondria ( 2 cells at left). Apoptotic cells at varying stages of mitochondrial DY appear green (3 cells at right)

 


Flow Cytometry                                                                              

 
Cells were analysed in a Becton Dickinson FACS Caliber Flow Cytometer.  Jurkat cells were treated with either DMSO (negative, non-induced cells) or with staurosporine (apoptotic; induced cells) for 3 hours at 370C,then labeled with MitoPT for 15 minutes.  Collapse of the mitochondrial
DY is indicated by an increase in the number of cells falling into R3 corresponding to a loss of red fluorescence, indicative of the onset of apoptosis.  In these figures of flow cytometry data, non-apoptotic cells are in R2, which prints in  green, and apoptotic cells are in R3, which prints in pink.

                                                                                                        

Mitochondrial Membrane Permeability Transition Detection Products

MitoPTTM (red and green fluorescence)

 

Product No. of Vials  Size Catalogue Number
MitoPT-JC1 1 100 test vial BB924
MitoPT-JC1 1 400 test vial BB911

For Research Use Only

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Metachem Diagnostics Ltd.
29 Forest Road, Piddington, Northampton. NN7 2DA
Tel. (01604) 870370     Fax (01604) 870194