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JC10 Cell Technology Mitochondria Membrane Potential
JC10
Flow Cytometry Mitochondrial Membrane Potential Detection Kit

$295.00

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Product code: JC10FC
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Product Description

Introduction

The loss of mitochondrial membrane potential is a hallmark for apoptosis.The APO LOGIX JC-10 Assay Kit measures the mitochondrial membrane potential in cells. In non-apoptotic cells, JC-10 exists as a monomer in the cytosol (green) and also accumulates as aggregates in the mitochondria which stain greenish orange. Whereas, in apoptotic and necrotic cells, JC-10 exists in monomeric form and stains the cytosol green. JC-10 was developed to increase solubality in aqueous buffers as compaired to its conterpart JC-1.

Key Benefits

  • Superior solubility in aqueous buffers as compared to JC-1.
  • Applications – Cell permeable no wash Flow Cytometry assay.
  • Add reagent directly to live cells, incubate 30-60 minutes and measure.

mitocondril_membran_a_b_1-1
Fig (A).
Jurkat cells were cultured with DMSO for 2 hours. Cells were then stained with JC-10 Mitochondrial Membrane Potential Detection Kit for 15 minutes and analyzed by flow cytometry.

mitocondril_membran_a_b_2_1
Fig (B).
Jurkat cells were cultured with staurosporine for 2 hours. Cell were then stained with JC-10 Mitochondrial Membrane Potential Detection Kit for 15 minutes and analyzed by flow cytometry.

Background

The mitochondrial permeability transition is an important step in the induction of cellular apoptosis. During this process, the electrochemical gradient (referred to as DY) across the mitochondrial membrane collapses. The collapse is thought to occur through the formation of pores in the mitochondria by dimerized Bax or activated Bid, Bak, or Bad proteins. Activation of these pro-apoptotic proteins is accompanied by the release of cytochrome c into the cytoplasm (1-4).

JC-1 has been used extensively to detect mitochondrial membrane potential, however due to its poor solubility in aqueous buffers it proves difficult to work with particularly when high concentrations are needed.

The JC-10 Assay Kit uses a unique cationic dye to signal the loss of the mitochondrial membrane potential. In healthy cells, the dye stains the mitochondria bright red. The negative charge established by the intact mitochondrial membrane potential allows the lipophilic dye, bearing a delocalized positive charge, to enter the mitochondrial matrix where it accumulates. When the critical concentration is exceeded, J-aggregates form which become fluorescent grennish orange where as it exists in the cytoplasm as monomers and fluoresces green. In apoptotic cells, the mitochondrial membrane potential collapses, and the JC-10 cannot accumulate within the mitochondria.

In these cells JC-10 remains in the cytoplasm in its monomeric green fluorescent form. Apoptotic cells, showing primarily green fluorescence, are easily differentiated from healthy cells which show grennish orange and green fluorescence. Both apoptotic and healthy cells can be visualized simultaneously by flow cytometry using 490 excitation and emission measured in FL1 (green) and FL2 (grennish orange). The JC-10 reagent is easy to use, simply dilute the reagent in the buffer provided or cell culture media and add it to the cells. After a 30-60- minute incubation analyze the samples via flow cytometry.

References

  • Desagher, S., Osen-Sand, A., Nichols, A., Eskes, R., Montessuit, S., Lauper, S., Maundrell, K., Antonsson, B., and Martinou, J.C. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144 (5): 891-901 (1999).
  • Narita, M., Shimizu, S., Ito, T., Chittenden, T., Lutz, R. J., Matsuda, H., and Tsujimoto, Y. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria. Proc. Natl. Acad. Sci. USA 95: 14681-14686 (1998).
  • Basanez, G., Nechushtan, A., Drozhinin, O., Chanturiya, A., Choe, E., Tutt, S., Wood, K. A., Hsu, Y. T., Zimmerberg, J., and Youle, R. J. Bax , but not Bcl-XL decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. USA 96: 5492-5497 (1999).
  • Luo, X., Budihardio, I., Zou, H., Slaughter, C., and Wang, X. Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490 (1998).

Kit contents and Long Term storage

ReagentPart#Temperature
JC-10 Dye ready to use in DMSO buffer at 2mg/mL (appox 3mM) 100mLPart# 4027Store at -20C
10X Assay BufferPart# 3002Store at 2-8C
FCCP Positive Control (1 vial)Part# 7025Store at -20C
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