Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Microplate Assays*
Although JC-1 is widely used in many labs, its poor water solubility makes it hard to use for some applications. Even at 1 µM concentration, JC-1 tends to precipitate in aqueous buffer. JC-10 has been developed to be a superior alternative to JC-1 where high dye concentration is desired. Compared to JC-1, our JC-10 has much better water solubility. JC-10 is capable of entering selectively into mitochondria, and changes reversibly its color from green to orange as membrane potentials increase. This property is due to the reversible formation of JC-10 aggregates upon membrane polarization that causes shifts in emitted light from 520 nm (i.e., emission of JC-10 monomeric form) to 570 nm (i.e., emission of J-aggregate). When excited at 490 nm, the color of JC-10 changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized. This Cell Meter™ JC-10 Mitochondrial Membrane Potential Assay Kit enable you to monitor mitochondrial membrane potential changes using a simple microplate reader while all the other commercial JC-1 assay kits require the use of a flow cytometer. Our kit provides the most robust method to monitor mitochondrial membrane potential changes, and can be readily used for screening a large compound library.
Example protocol
AT A GLANCE
Protocol summary
- Prepare cells
- Add test compounds
- Add JC-10 dye-working solution (50 µL/well/96-well plate or 12.5 µL/well/384-well plate)
- Incubate at 37°C, 5% CO2 incubator for 30 to 60 minutes
- Add Assay Buffer B (50 µL/well/96-well plate or 12.5 µL/well/384-well plate)
- Monitor fluorescence intensities (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) and 540/590 nm (Cutoff = 570 nm)
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Add 50 µL of 100X JC-10 (Component A) into 5 mL of Assay Buffer A (Component B) and mix well to make JC-10 dye-working solution. Protect from light.
For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
SAMPLE EXPERIMENTAL PROTOCOL
- Treat cells by adding 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-plate) into the desired buffer (such as PBS or HHBS). Note: It is not necessary to wash cells before adding compound. However, if tested compounds are serum sensitive, growth medium and serum factors can be aspirated away before adding compounds. Add the same volume of HHBS into the wells (such as 90 µL for a 96-well plate or 20 µL for a 384-well plate) after aspiration. Alternatively, cells can be grown in serum-free media.
- Incubate the cell plate at room temperature or in a 37°C, 5% CO2 incubator for at least 15 minutes or a desired period of time (for Jurkat cells, 4 - 6 hours with camptothecin or 3 - 5 hours with staurosporine treatment) to induce apoptosis.
- Add 50 µL/well (96-well plate) or 12.5 µL/well (384-well plate) of JC-10 dye-working solution into the cell plate.
- Incubate the plate in a 37°C, 5% CO2 incubator for 30 - 60 minutes, protected from light. Note: The appropriate incubation time depends on the individual cell type and cell concentration used. Optimize the incubation time for each experiment.
- Add 50 µL/well (96-well plate) or 12.5 µL/well (384-well plate) of Assay Buffer B (Component C) into JC-10 dye-working solution plate before reading the fluorescence intensity. Note: DO NOT wash the cells after loading. For non-adherent cells, it is recommended to centrifuge cell plates at 800 rpm for 2 minutes with brake off after adding Assay Buffer B (Component C).
- Monitor the fluorescence intensities with a fluorescence microplate reader (bottom read mode) at Ex/Em = 490/525 nm (Cutoff = 515 nm) and 540/590 nm (Cutoff = 570 nm) for ratio analysis.
Spectrum
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Citations
View all 98 citations: Citation Explorer
Astilbin Induces Apoptosis in Oral Squamous Cell Carcinoma through p53 Reactivation and Mdm-2 Inhibition
Authors: Wu, Aimin and Zhao, Chungang
Journal: (2024): 1--13
Authors: Wu, Aimin and Zhao, Chungang
Journal: (2024): 1--13
Release of mitochondrial dsRNA into the cytosol is a key driver of the inflammatory phenotype of senescent cells
Authors: L{\'o}pez-Polo, Vanessa and Maus, Mate and Zacharioudakis, Emmanouil and Lafarga, Miguel and Attolini, Camille Stephan-Otto and Marques, Francisco DM and Kovatcheva, Marta and Gavathiotis, Evripidis and Serrano, Manuel
Journal: Nature Communications (2024): 7378
Authors: L{\'o}pez-Polo, Vanessa and Maus, Mate and Zacharioudakis, Emmanouil and Lafarga, Miguel and Attolini, Camille Stephan-Otto and Marques, Francisco DM and Kovatcheva, Marta and Gavathiotis, Evripidis and Serrano, Manuel
Journal: Nature Communications (2024): 7378
A non-Bactericidal Cathelicidin with Antioxidant Properties Ameliorates UVB-Induced Mouse Skin Photoaging via Intracellular ROS Scavenging and Keap1/Nrf2 Pathway Activation
Authors: Feng, Guizhu and Chen, Qian and Liu, Jin and Li, Junyu and Li, Xiang and Ye, Ziyi and Wu, Jing and Yang, Hailong and Mu, Lixian
Journal: Free Radical Biology and Medicine (2024)
Authors: Feng, Guizhu and Chen, Qian and Liu, Jin and Li, Junyu and Li, Xiang and Ye, Ziyi and Wu, Jing and Yang, Hailong and Mu, Lixian
Journal: Free Radical Biology and Medicine (2024)
Blast-overpressure induced modulation of PARP-SIRT-NRF2 axis in stress signaling of astrocytes and microglia.
Authors: MUTHAIAH, VIJAYA PRAKASH KRISHNAN
Journal: (2024)
Authors: MUTHAIAH, VIJAYA PRAKASH KRISHNAN
Journal: (2024)
Safe transportation and targeted destruction: Albumin encapsulated aggregation-induced emission photosensitizer nanoaggregate for tumor photodynamic therapy through mitochondria damage-triggered pyroptosis
Authors: Cao, Juanmei and Qu, Yong and Zhu, Shaojie and Zhan, Jinshan and Xu, Yiting and Jin, Yifan and Wang, Yuqing and Li, Zhuoxia and Chai, Chuxing and Wu, Xiangwei and others,
Journal: Aggregate (2024): e637
Authors: Cao, Juanmei and Qu, Yong and Zhu, Shaojie and Zhan, Jinshan and Xu, Yiting and Jin, Yifan and Wang, Yuqing and Li, Zhuoxia and Chai, Chuxing and Wu, Xiangwei and others,
Journal: Aggregate (2024): e637
Page updated on October 31, 2024