05 Cell Staining

JC-1 MitoMP Detection Kit

JC-1 MitoMP Detection Kit

Mitochondrial Membrane Potential Detection

  • Product code
    MT09  JC-1 MitoMP Detection Kit
Unit size Price Item Code
10 set $242.00 MT09-10
ORDER
Component
10 set JC-1 Dye
Imaging Buffer (10x)
100 nmol x1
11 ml x1

Description

Mitochondria synthesize ATP using oxygen to produce necessary energy for living cells. Lowering of mitochondrial activity and dysfunction are known to be closely related to cancer, aging, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases. Mitochondrial membrane potential is a parameter used to measure with mitochondrial condition.

 

Manual

Technical info

JC-1 forms aggregate (in healthy mitochondria) with red fluorescence. As membrane potential decreases, JC-1 becomes monomers, which shows in green fluorescence. The change in ratio of red to green fluorescence is used as a indicator of mitochondrial condition.

Easy to Use

Easy to dissolve

JC-1 has been difficult to dissolve, but this kit solves the problem!

Detect by Several Equipments

Please refer to Data: Induced Apoptosis for experimental examples

Imaging Buffer Included
HEPES included Imaging Buffer keeps the cell condition optimal for a long period

Procedure

Data: Depolarization

HeLa cells treated with depolarizing reagent, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) were stained with JC-1 MitoMP Detection Kit. Red fluorescence indicates normal membrane potential or health mitochondria. Untreated cells showed red fluorescence, while FCCP treated cells showed little red fluorescence.

Experimental Condition
JC-1 concentration:
2 μmol/l in MEM, staining time: 30 min
FCCP concentration:
100 μmol/l, FCCP treatment time: 1 h

Imaging Condition
Green: Ex 488 nm / Em 500-550 nm
Red: Ex 561 nm / Em 560-610 nm
Scale Bar: 20 μm

Data: Induced Apoptosis

Jurkat cells treated by apoptosis inducing reagent, Staurosporine, were stained with JC-1 MitoMP Detection Kit. Procedures for these data can be found in the Technical Manual.

[Fluorescence Microscope]
Fluorescence imaging of mitochondrial membrane potential in Jurkat cells

Imaging Condition

Green: Ex 488 nm / Em 500-550 nm
Red: Ex 561 nm / Em 560-610 nm
Scale Bar: 80 μm

[Flow Cytometry]
Flow cytometric analysis of mitochondrial membrane potential in Jurkat cells

Detecting Condition

Green: Ex 488 nm / Em 515-545 nm
Red: Ex 488 nm / Em 564-604 nm

Plate Reader
Fluorescence intensity ratio of mitochondrial membrane potential in Jurkat cells

Detecting Condition

Green: Ex 485 nm / Em 525-545 nm
Red: Ex 535 nm / Em 585-605 nm

Required amount of Imaging Buffer solution by vessel type

Mitophagy Induction and Detection of Mitochondrial Membrane Potential Changes

Mitochondrial condition in the carbonyl cyanide m-chlorophenyl hydrazine (CCCP) treated Parkin-expressing HeLa cells was compared with untreated cells using Mitophagy Detection Kit (MD01, MT02) and JC-1 MitoMP Detection Kit (MT09).

Result:
Mitophagy was not detected in untreated cells and the membrane potential was normal. However, reduction of membrane potential and mitophagy were observed in treated cells.

Experimental Condition

Transfection of Parkin plasmid to HeLa cells
・HileyMax (H357) was used to transfect Parkin plasmid to HeLa cells (Parkin plasmid/HilyMax reagent: 0.1μg/0.2 μL) by incubating overnight.

Detection of Mitophagy
1. Add 0.1 μmol/L Mtphagy working solution to Parkin expressing HeLa cells and incubated for 30 minutes at 37 ℃
2. Wash cells with HBSS
3. Add 10 μg/mL CCCP/MEM solution and inclubate for 2 hours at 37 ℃
4. Observe under fluorescence microscope

Detection of Mitochondrial Membrane Potential
1. Add 10 μg/mL CCCP/MEM solution to Parkin expressing HeLa cells and incubate for 1.5 hours at 37 ℃
2. Add 4 μmol/L JC-1 working solution (final concentration: 2 μmol/L) and incubate for 30 minutes at 37 ℃
3. Wash with HBSS and add Imaging Buffer Solution.
4. Observe under fluorescence microscope

Detecting Condition

[Mitophagy Detection] 

Ex: 561 nm, Em: 570-700 nm

[Mitochondrial Membrane Potential Detection]

Green Ex: 488 nm, Em: 500-550 nm
Red Ex: 561 nm, Em: 560-610 nm

References

Open References

No. Sample Instrument Reference (Link)
1) Cell
(U2OS, HeLa)
Fluorescent Microscope T. Namba, "BAP31 regulates mitochondrial function via interaction with Tom40 within ER-mitochondria contact sites ", Sci Adv., 2019, 5, (6), 1386.
2) Cell
(Neuron)
Fluorescent Microscope I. Kawahata, L. Luc Bousset, R. Melki and K. Fukunaga, "Fatty Acid-Binding Protein 3 is Critical for α-Synuclein Uptake and MPP+-Induced Mitochondrial Dysfunction in Cultured Dopaminergic Neurons ", Int J Mol Sci., 2019, 20, 5358.
3) Cell
(3T3L1, C2C12)
Plate Reader M. Kurano, K. Tsukamoto, T. Shimizu, H. Kassai, K. Nakao, A. Aiba, M. Hara and Yatomi, "Protection Against Insulin Resistance by Apolipoprotein M/Sphingosine 1-Phosphate ", Diabetes, 2020, DOI: 10.2337/db19-0811.
4) Cell
(ALM)
Plate Reader T. Nechiporuk, S.E. Kurtz, O. Nikolova, T. Liu, C.L. Jones, A. D. Alessandro, R. C. Hill, A. Almeida, S. K. Joshi, M. Rosenberg, C. E. Tognon, A. V. Danilov, B. J. Druker, B. H. Chang, S. K McWeeney and J. W. Tyner, "The TP53 Apoptotic Network Is a Primary Mediator of Resistance to BCL2 Inhibition in AML Cells.", Cancer Discov, 2019, 9,
5) Cell
(Macrophage)
Fluorescent Microscope G. Yang, M. Fan, J. Zhu, C. Ling, L. Wu, X. Zhang, M. Zhang, J. Li, Q. Yao, Z. Gu and X. Cai, "A multifunctional anti-inflammatory drug that can specifically target activated macrophages  massively deplete intracellular H2Oand produce large amounts CO for a highly efficient treatment of osreoarthritis"Biomaterials2020,  doi:10.1016/j.biomaterials.2020.120155.
6) Cell
(ARPE-19)
Fluorescent Microscope J. H. Quan, F. F. Gao, H. A. Ismail, J. M.  Yuk, G. H. Cha, J. Q. Chu and Y. H. Lee,  "Silver Nanoparticle-Induced Apoptosis in ARPE-19 Cells Is Inhibited by Toxoplasma gondii Pre-Infection Through Suppression of NOX4-Dependent ROS Generation"Int J Nanomedicine2020, 15, 3695–3716.
7) Cell
(A549)
Flow Cytometer C. N. D’Alessandro-Gabazza, T. Yasuma, T. Kobayashi, M. Toda1, A. M. Abdel-Hamid, H. Fujimoto, O. Hataji, H. Nakahara, A. Takeshita, K. Nishihama, T. Okano, H. Saiki, Y. Okano, A. Tomaru, V. F. D’Alessandro, M. Shiraishi, A. Mizoguchi, R. Ono, J. Ohtsuka, M. Fukumura, T. Nosaka, X. Mi, D. Shukla, K. Kataoka, Y. Kondoh, M. Hirose, T. Arai, Y. Inoue, Y. Yano, R. I. Mackie, I. Cann and E. C. Gabazza, "Inhibition of lung microbiota-derived proapoptotic peptides ameliorates acute exacerbation of pulmonary fibrosis", Nat. Comm.2022, doi:10.1038/s41467-022-29064-3.

Handling and storage condition

Handling and storage condition
0-5°C
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