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Cell Meter™ JC-10 Mitochondrion Membrane Potential Assay Kit *Optimized for Microplate Assays*

Campotothecin-induced mitochondria membrane potential changes were measured with JC-10™ and JC-1 in Jurkat cells. After Jurkat cells were treated with camptothecin (10 µM) for 4 hours, JC-1 and JC-10™ dye working solutions were added to the wells and incubated for 30 minutes. The fluorescence intensities for both J-aggregates (highlighted in blue) and monomeric forms (highlighted in red) of JC-1 and JC-10™ were measured at Ex/Em = 490/525 nm (Cutoff = 515 nm) and 490/590 nm  (Cutoff = 570 nm) with NOVOstar microplate reader (BMG Labtech).
Campotothecin-induced mitochondria membrane potential changes were measured with JC-10™ and JC-1 in Jurkat cells. After Jurkat cells were treated with camptothecin (10 µM) for 4 hours, JC-1 and JC-10™ dye working solutions were added to the wells and incubated for 30 minutes. The fluorescence intensities for both J-aggregates (highlighted in blue) and monomeric forms (highlighted in red) of JC-1 and JC-10™ were measured at Ex/Em = 490/525 nm (Cutoff = 515 nm) and 490/590 nm  (Cutoff = 570 nm) with NOVOstar microplate reader (BMG Labtech).
Ordering information
Price ()
Catalog Number22800
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Spectral properties
Excitation (nm)508
Emission (nm)524
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Excitation (nm)
508
Emission (nm)
524
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.

Platform


Fluorescence microplate reader

Excitation490/540 nm
Emission525/590 nm
Cutoff515/570 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode

Components


Component A: 100X JC-10 in DMSO1 vial (250 µL)
Component B: Assay Buffer A1 bottle (25 mL)
Component C: Assay Buffer B1 bottle (25 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells
  2. Add test compounds
  3. Add JC-10 dye-working solution (50 µL/well/96-well plate or 12.5 µL/well/384-well plate) 
  4. Incubate at 37°C, 5% CO2 incubator for 30 to 60 minutes
  5. Add Assay Buffer B (50 µL/well/96-well plate or 12.5 µL/well/384-well plate)
  6. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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).

  6. 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


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)508
Emission (nm)524

Citations


View all 60 citations: Citation Explorer
Impact of Organelle Transport Deficits on Mitophagy and Autophagy in Niemann--Pick Disease Type C
Authors: Liedtke, Maik and V{\"o}lkner, Christin and Hermann, Andreas and Frech, Moritz J
Journal: Cells (2022): 507
Antidepressants induce toxicity in human placental BeWo cells
Authors: Nabekura, Tomohiro and Ishikawa, Shinya and Tanase, Makoto and Okumura, Taichi and Kawasaki, Tatsuya
Journal: Current Research in Toxicology (2022): 100073
Mitochondria Transfer from Adipose Stem Cells Improves the Developmental Potential of Cryopreserved Oocytes
Authors: Kankanam Gamage, Udayanga Sanath and Hashimoto, Shu and Miyamoto, Yuki and Nakano, Tatsuya and Yamanaka, Masaya and Koike, Akiko and Satoh, Manabu and Morimoto, Yoshiharu
Journal: Biomolecules (2022): 1008
Patchouli alcohol suppresses castration-resistant prostate cancer progression by inhibiting NF-$\kappa$B signal pathways
Authors: Cai, Jian and Zhao, Juan and Gao, Ping and Xia, Yuguo
Journal: Translational Andrology and Urology (2022): 528
DNA repair proteins as the targets for paroxetine to induce cytotoxicity in gastric cancer cell AGS
Authors: Liu, Bang-Hung and Yuan, Tein-Ming and Huang, Chih-Jou and Hsu, Duan-Ting and Chen, Shi-Wen and Hsiao, Nai-Wan and Lin, Sheng-Chih and Wu, Shu-Wan and Lin, Yi-Mei J and Chuang, Show-Mei
Journal: American journal of cancer research (2022): 1465
Effects of time-restricted feeding and type of food on fertility competence in female mice
Authors: Konishi, Nafuko and Matsumoto, Hiroshi and Hashimoto, Shu and Gamage, Udayanga Sanath Kankanam and Tachibana, Daisuke and Fukuda, Aisaku and Morimoto, Yoshiharu and Koyama, Masayasu
Journal: Scientific Reports (2022): 1--15
The relationship between free fatty acids and mitochondrial oxidative stress damage to trophoblast cell in preeclampsia
Authors: Jiang, Lingling and Yan, Jianying
Journal: BMC Pregnancy and Childbirth (2022): 1--6
Antineoplastic Effects and Mechanisms of a New RGD Chimeric Peptide from Bullfrog Skin on the Proliferation and Apoptosis of B16F10 Cells
Authors: Jiang, Xuan and Zhang, Xin and Fu, Chao and Zhao, Ruili and Jin, Tianming and Liu, Mengyue and Pan, Chenhao and Li, Liu An and Ma, Jifei and Yu, Enyuan and others,
Journal: The Protein Journal (2021): 1--12
Human VAMP3 Suppresses or Negatively Regulates Bax Induced Apoptosis in Yeast
Authors: Akintade, Damilare D and Chaudhuri, Bhabatosh
Journal: Biomedicines (2021): 95