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JC-10
JC-10
DESCRIPTION:
Name：The JC-10 chemical structure is undisclosed. Molecular Weight: ~ 600； Solvents: soluble in DMSO
Introduction:
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 an 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. 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. We have also developed a protocol to use JC-10 in fluorescence microplate platform. In some cell lines JC-10 has even superior performance to JC-1.
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-10 working solution:
1.1 Prepare a 2 to 10 mM stock solution of JC-10 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 Prepare a 1X JC-10 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-10 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-10 working solution (from step 1.2) into the cell plate.
2.3 Incubate the JC-10 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-10 working solution from the plate, wash the cells with HHBS or buffer of your choice. 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-10 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-10 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
4.1 Set the fluorescent plate reader to perform an endpoint read.
4.2 Set excitation wavelength at 535 ± 17.5nm (aggregate excitation only) or at 475 ± 20nm (for simultaneous aggregate and monomer excitation)
4.3 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.
4.4 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 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 signal may indicate either mitochondrial depolarization or cell death and must be interpreted in parallel with a cytotoxicity assay.
NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.
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Description
| Name | JC-10 | ||
|---|---|---|---|
| CAT# | 22204 | CAS# | N/A |
| Storage# | -20°C Sealed & desiccated & Minimized light exposure & Minimized Freezing And Thawing! | Shelf Life# | 24 months |
| Ex(nm)# | 510 | Em(nm)# | 525 |
| MW# | 600 | Solvent# | DMSO |
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