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

Effect of FCCP induced mitochondria membrane potential change in Jurkat cells. Jurkat cells were dye loaded with JC-10 dye working solution along with DMSO (Top) or 5 µM FCCP (Low) for 10 minutes. The fluorescence intensities for both J-aggregates and monomeric forms of JC-10 were measured with a FACSCalibur (Becton Dickinson) flow cytometer using FL1 and FL2 channels. Uncompensated data (left column) were compared with compensated data (right column).
Effect of FCCP induced mitochondria membrane potential change in Jurkat cells. Jurkat cells were dye loaded with JC-10 dye working solution along with DMSO (Top) or 5 µM FCCP (Low) for 10 minutes. The fluorescence intensities for both J-aggregates and monomeric forms of JC-10 were measured with a FACSCalibur (Becton Dickinson) flow cytometer using FL1 and FL2 channels. Uncompensated data (left column) were compared with compensated data (right column).
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Price ()
Catalog Number22801
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 causes extraordinary inconvenience. Even at 1 µM concentration, JC-1 tends to precipitate in aqueous buffer. JC-10 is developed to be a superior alternative to JC-1 where high dye concentration is desired. Compared to JC-1, 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. Both colors can be detected using the filters commonly mounted in all flow cytometers, so that green emission can be analyzed in fluorescence channel 1 (FL1) and greenish orange emission in channel 2 (FL2). Besides its potential use in flow cytometry, it can also be used in fluorescence imaging and fluorescence microplate platform. This kit provides all the essential components with an optimized assay method for the detection of apoptosis in cells with the loss of mitochondrial membrane potential. This fluorometric assay is based on the detection of the mitochondrial membrane potential changes in cells by the cationic, lipophilic JC-10 dye. In normal cells, JC-10 concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. However, in apoptotic and necrotic cells, JC-10 exists in monomeric form and stains cells in green fluorescence. The kit is optimized for screening of apoptosis activators and inhibitors by flow cytometry. We also offer a convenient 96-well and 384-well fluorescence microtiter-plate format kit (cat#22800) for high through put screening.

Platform


Flow cytometer

Excitation488 nm laser
Emission530/30 nm, 575/26 nm filter
Instrument specification(s)FITC, PE channel

Components


Component A: 200X JC-10 in DMSO1 vial (250 µL)
Component B: Assay Buffer1 bottle (50 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare cells with test compounds at the density of 5 × 105 to 1 x 106 cells/mL
  2. Resuspend the cells in 500 µL of JC-10 working solution (2-5 × 105 cells/tube) 
  3. Incubate at 37°C or room temperature for 15-60 minutes
  4. Analyze with flow cytometer using FL1 channel (green fluorescence monomeric signal) and FL2 channel (orange fluorescence aggregated signal)

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

Add 25 µL of 200X JC-10 (Component A) into 5 mL of Assay Buffer A (Component B) and mix well to make JC-10 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 with test compounds for a desired period of time to induce apoptosis. Set up parallel control experiments.

    For Negative Control: Treat cells with vehicle only.

    For Positive Control: Treat cells with FCCP or CCCP at 2-10  µM in a 37 oC, 5% CO2 incubator for 15 to 30 minutes. Note: CCCP or FCCP can be added simultaneously with JC-10 working solution. Titration of the CCCP or FCCP may be required for optimal results with an individual cell lines. 

  2. Centrifuge the cells to get 2 - 5 × 105 cells per tube. Note: For adherent cells, gently lift the cells by 0.5 mM EDTA to remain the cells intake, and wash the cells once with serum-containing media prior to incubation with JC-10 working solution.

  3. Resuspend cells in 500 µL of JC-10 working solution.

  4. Incubate the cells at room temperature or in a 37 oC, 5% CO2 incubator for 15 - 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. Monitor the fluorescence intensity using flow cytometer with FL1 channel for the green fluorescence monomeric signal (in apoptotic cells), and FL2 channel for the orange fluorescence aggregated signal (in healthy cells). Gate on the cells, excluding debris. It is recommended that compensation corrections be performed using the FCCP or CCCP-treated cells.
  • Typical Flow Cytometer settings for the analysis of JC-10 on a BD FACS Calibur System flow cytometer are as follows:
    Suggested initial conditions may require modifications because of differences in cell types and culture conditions, and also the individual instrumentation.
    FL1 PMT voltage 366 
    FL2 PMT voltage 430
    Compensation: FL1 – 47.2% FL2; FL2 – 47.0% FL1

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)508
Emission (nm)524

Citations


View all 52 citations: Citation Explorer
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
Gracillin Isolated from Reineckia carnea Induces Apoptosis of A549 Cells via the Mitochondrial Pathway
Authors: Yang, Jianqiong and Cao, Ling and Li, Yamei and Liu, Hai and Zhang, Minhong and Ma, Huamou and Wang, Biao and Yuan, Xiaoliang and Liu, Qian
Journal: Drug design, development and therapy (2021): 233
Mitochondrial Function and Root-Filled Teeth--Detrimental and Unknown Interfaces in Systemic Immune Diseases
Authors: Lechner, Johann and Mayer, Wolfgang
Journal: International Journal of General Medicine (2020): 387--402
Inhibition of phosphodiesterase-10A by Papaverine protects human cortical neurons from quinolinic acid induced oxidative stress and synaptic proteins alterations
Authors: Bhat, Abid and Tan, Vanessa and Heng, Benjamin and Essa, Musthafa M and Chidambaram, Saravana B and Guillemin, Gilles J
Journal: bioRxiv (2020)
Roflumilast, a cAMP-Specific Phosphodiesterase-4 Inhibitor, Reduces Oxidative Stress and Improves Synapse Functions in Human Cortical Neurons Exposed to the Excitotoxin Quinolinic Acid
Authors: Bhat, Abid and Tan, Vanessa and Heng, Benjamin and Lovejoy, David B and Sakharkar, Meena Kishore and Essa, Musthafa Mohamed and Chidambaram, Saravana Babu and Guillemin, Gilles J
Journal: ACS Chemical Neuroscience (2020)
Activation of TFEB-mediated autophagy by trehalose attenuates mitochondrial dysfunction in cisplatin-induced acute kidney injury
Authors: Zhu, Lingling and Yuan, Yujia and Yuan, Longhui and Li, Lan and Liu, Fei and Liu, Jingping and Chen, Younan and Lu, Yanrong and Cheng, Jingqiu
Journal: Theranostics (2020): 5829
Genome-wide R-loop Landscapes during Cell Differentiation and Reprogramming
Authors: Yan, Pengze and Liu, Zunpeng and Song, Moshi and Wu, Zeming and Xu, Wei and Li, Kuan and Ji, Qianzhao and Wang, Si and Liu, Xiaoqian and Yan, Kaowen and others,
Journal: Cell Reports (2020): 107870
Curcumin induces apoptosis in lung cancer cells by 14-3-3 protein-mediated activation of Bad
Authors: Endo, Hiroshi and Inoue, Izumi and Masunaka, Kimiko and Tanaka, Masaya and Yano, Mihiro
Journal: Bioscience, Biotechnology, and Biochemistry (2020): 2440--2447
A Preclinical Evaluation towards the Clinical Application of Oxygen Consumption Measurement by CERMs by a Mouse Chimera Model
Authors: Kuno, Takashi and Tachibana, Masahito and Fujimine-Sato, Ayako and Fue, Misaki and Higashi, Keiko and Takahashi, Aiko and Kurosawa, Hiroki and Nishio, Keisuke and Shiga, Naomi and Watanabe, Zen and others,
Journal: International journal of molecular sciences (2019): 5650
A novel hiPSC-derived system for hematoendothelial and myeloid blood toxicity screens identifies compounds promoting and inhibiting endothelial-to-hematopoietic transition
Authors: Elcheva, Irina and Sneed, Mechelle and Frazee, Scott and Liu, Zhenqiu and Zhu, Junjia and Wood, Tyler and Hendrickson, Sara and Oehler, Chuck and Garcia, Brad and Spiegelman, Vladimir S
Journal: Toxicology in Vitro (2019): 104622