中文 | EN
JC-1
JC-1
CAS#：3520-43-2；
Chemical Name：JC-1 [5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide]
Introduction:
JC-1 (5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine chloride) is a mitochondrial dye that stains mitochondria in living cells in a membrane potential-dependent fashion.JC-1 monomer is in equilibrium with so-called J-aggregates, which are favored at higher dye concentration or higher mitochondrial membrane potential. At low concentrations (due to low ∆ψm) JC-1 is predominantly a JC-1 monomer that yields green fluorescence with emission of 530±15nm. At high concentrations (due to high ∆ψm) the dye J-aggregates yielding a red to orange colored emission (590±17.5nm). JC-1 is suitable for the labeling of mitochondria in live cells and is not compatible with fixation. Therefore a decrease in the aggregate fluorescent count is indicative of depolarization whereas an increase is indicative of hyperpolarization, then we can use the fluorescence ratio technique to study mitochondrial membrane potentials.
Membrane potential (∆ψm) is highly interlinked to many mitochondrial processes. The ∆ψm controls ATP synthesis, generation of ROS, mitochondrial calcium sequestration, import of proteins into the mitochondrion and mitochondrial membrane dynamics. Conversely, ∆ψm is controlled by ATP utilization, mitochondrial proton conductance, respiratory chain capacity and mitochondrial calcium. Hence pharmacological changes in ∆ψm can be associated with a multitude of other mitochondrial pathological parameters which may require further independent evaluation.
Depolarization can be found in the presence of ionophores that could induce nonselective cation channels or become selective mobile ionic carriers. Protonophores such as FCCP and CCCP induce reversal of the ATPase, as a compensatory mechanism that tries to maintain ∆ψm, which will deplete ATP even in the presence of a normal glycolytic pathway. Hyperpolarization could be found in the presence of ATPase inhibition, inadequate supply of ADP, increased supply of NADH, apoptosis due to oxidative stress and potentially proton slippage due to cytochrome c oxidase dephosphorylation. In either scenario, OXPHOS uncoupling ensues
JC-1 is particularly useful for apoptosis studies. In apoptotic cells, the dye stays in the cytoplasm and fluoresces green. It has also been used in high throughput drug screening applications.
JC-1, iodide is the original salt form of the dye. We developed JC-1 chloride salt for applications where iodide ion is not desired. JC-1 chloride may also have the advantage of being slightly more water soluble than the iodide salt.
1 ε (MeOH) = 190,000
2 Red solid soluble in DMSO
3 Store at -20°C and protect from light, especially in solution
Protocol for measure the cell membrane potential
The following protocol is only a guideline for measurement of membrane potential to measure the cell membrane potential. Different cell lines will require optimization of protocol, in particular the JC-1 concentration and the cell seeding per well
1. Prepare JC-1 working solution:
1.1 Prepare a 2 to 10 mM stock solution of JC-1 in anhydrous DMSO. The stock solution should be freshly prepared just before use; The rest solution should be kept in < -20oC to preserve, Avoid repeated freeze-thaw cycles
1.2 Freshly prepare a 1X JC-1 working solution: Just before the experiment, either dissolve JC-1 solid in DMSO or thaw an aliquot of the JC-1 stock solution to room temperature. Prepare a 1X working solution of 10 to 30 µM in Hanks and 20 mM Hepes buffer (HHBS) or other buffer such as PBS, pH 7 with 0.02% PF-127. Mix them well by votexing.
2. Run JC-1 assay with a fluorescence microplate reader
2.1 Treat cells with test compounds for appropriate time (For example, Jurkat cells can be treated with camptothecin for 4-6 hours) to induce apoptosis. For blank wells (medium without the cells), add the corresponding amount of compound buffer.
2.2 Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of JC-1 working solution (from step 1.2) into the cell plate.
2.3 Incubate the JC-1 loading plate at 37oC, 5% CO2 for 15-60 min, protecting it from the light.
Note 1: The incubation time and cell concentration should be optimized according to the cell type.
2.4 Remove the JC-1 working solution from the plate, wash the cells with HHBS or other buffer such as PBS. Add 100 µL/well (96-well plate) or 25 µL/well (384-well plate) of HHBS back to the cell plate.
2.5 Monitor the fluorescence at Emission 525 and 590 nm (excitation: 490-500 nm) for ratio analysis.
3. Run JC-1 assay with a fluorescence microscope or a flow cytometer
3.1 Treat cells with test compounds for appropriate time (For example, Jurkat cells can be treated with camptothecin for 4-6 hours) to induce apoptosis.
3.2 Centrifuge the cells to get 1-5 ×105 cells per tube.
3.3 Resuspend cells in 500 µL of JC-1 working solution (from step 1.2).
3.4 Incubate at room temperature or 37°C for 10 to 30 min, protecting it from the light.
3.5 Wash the cells with HHBS or buffer of your choice. Resuspend cells in 500 µL of HHBS to get 1-5 ×105cells per tube.
3.6 Monitor the fluorescence at Ex/Em = 490/525 and 590 nm with a fluorescence microscope (using FITCand TRITC filters) or a flow cytometer (using FL1 and FL2 channels).
4. Fluorescence Reading
1. Set the fluorescent plate reader to perform an endpoint read.
2. Set excitation wavelength at 535 ± 17.5nm (aggregate excitation only) or at 475 ± 20nm (for simultaneous aggregate and monomer excitation)
3. Set emission wavelength at 590 ± 17.5nm (aggregate emission only). If reading of the monomer species is also desired, set a second emission reading at 530 ± 15nm.
4. FCCP or CCCP 100μM treatment for 4 hours should decrease the JC- 1 aggregate signal to at least 25 – 30% from control levels.
5. Data Analysis and Sample Data
Subtract background (A590 of non-stained cells) from test signal and express as percentage from control. Data obtained with the JC-1 assay gives a relative measure of mitochondrial membrane potential as a percentage of control and cannot be used for absolute measurements of membrane potential in millivolts. Decrease in JC-1 signal may indicate either mitochondrial depolarization or cell death and must be interpreted in parallel with a cytotoxicity assay. The data in Figure 1 below shows the uncoupling effect of FCCP acute treatment on HL60 cells as measured with the JC-1 stain and read on a fluorescent plate reader.
NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.
注意事项：
JC-1如果一次使用量较小，需把每管再适当进行分装，尽量避免反复冻融。
为了您的安全和健康，请穿实验服并戴一次性手套操作。
本公司所有产品仅限用于研发，操作时必须戴口罩/手套/实验服和其它生化实验室防护措施。
产品参数
| Name | JC-1， [5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide] | ||
|---|---|---|---|
| CAT# | 22200 | CAS# | 3520-43-2; 47729-63-5 |
| Storage# | -20°C Sealed & desiccated & Minimized light exposure | Shelf Life# | 24 months |
| Ex(nm)# | 515 | Em(nm)# | 529 |
| MW# | 652.23 | Solvent# | DMSO |
The ultimate scientific research that strives for excellence is manifested in the details, forging the beauty of product quality.
Day after day, we persist in the pursuit of innovation. Through dedication, we experience and achieve a leading position in technology.
Room 208, 2nd Floor, Building 2, No. 4 Courtyard, Jinhang West Road, Shunyi District, Beijing
Customer Service hotline：400-686-3443
Email：info@fluorescence.cn