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线粒体膜电位检测试剂盒(JC-10)
JC-10 Mitochondrial Membrane Potential Detection Kit.
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 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.
Warnings and Precautions
1. For Research Use Only. Not for use in diagnostic procedures.
2. Gloves, protective clothing and eyewear should be worn and safe laboratory practices followed.
Kit Components:
|
Reagents |
Package |
|
JC-10(200×) in DMSO |
100μL/tube，5 tubes |
|
Pure Water |
90mL |
|
Assay Buffer (5×) |
80mL |
|
CCCP(10mM) |
20μL |
Storage and Shelf Life:
1. Store the kit at 2°C to 8°C until first use. The shelf time is 1 year
2. Reconstituted JC-1 reagent should be aliquoted in small amounts sufficient for one day of experimental work and stored at or below -20°C, protected from light and moisture (preferably in a desiccator). Shelf life of reconstituted JC-1 is six months if stored at or below -20°C. Avoid multiple freeze-thaw cycles
Protocol Optimized for Microplate Assays
1. Prepare cells:
1.1 For adherent cells: Plate cells overnight in growth medium at 20,000 to 80,000 cells/well/90µL for a 96-well plate or 5,000 to 20,000 cells/well/20µL for a 384-well plate.
1.2 For non-adherent cells: Centrifuge the cells from the culture medium and then suspend the cell pellet in culture medium at 100,000-200,000 cells/well/90 μL for a 96-well poly-D lysine plate or 25,000-50,000 cells/well/20 μL for a 384-well poly-D lysine plate. Centrifuge the plate at 800 rpm for 2 minutes with brake off prior to the experiments.
Note: Each cell line should be evaluated on an individual basis to determine the optimal cell density for apoptosis induction.
2. Prepare JC-10 dye-loading solution:
2.1 Thaw all the kit components at room temperature before use.
2.2 Add 50µL of 100X JC-10 (Component A) into 5 mL of Assay Buffer A (Component B), and mix well.
Note: Aliquot and store the unused 100X JC-10 (Component A) at -20 oC. Avoid repeated freeze/thaw cycles.
3. Run JC-10 assay:
3.1 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.
3.2 Incubate the cell plate at room temperature or in a 37 oC, 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.
3.3 Add 50 µL/well (96-well plate) or 12.5 µL/well (384-well plate) of JC-10 dye-loading solution (from step 2.2) into the cell plate (from Step 3.2).
3.4 Incubate the dye-loading plate in a 37 oC, 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.
3.5 Add 50 µL/well (96-well plate) or 12.5 µL/well (384-well plate) of Assay Buffer B (Component C) into the dye-loading plate (from Step 3.4) before reading the fluorescence intensity.
Note 1: DO NOT wash the cells after loading. 2: 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).
3.6 Monitor the fluorescence intensities at Ex/Em = 490/525 nm (cut off at 515 nm) and 540/590 nm (cut off at 570 nm) with bottom read mode for ratio analysis
Protocol Optimized for Flow Cytometry Assays
1. Prepare cells: Prepare cells at the density from 5 × 105 to 1 × 106 cells/mL.
Note: Each cell line should be evaluated on the individual basis to determine the optimal cell density for apoptosis induction.
2. Prepare JC-10 dye-loading solution:
2.1 Thaw all the kit components at room temperature before use.
2.2 Add 25µL of 200X JC-10 (Component A) into 5 mL of Assay Buffer (Component B), and mix well.
Note: Aliquot and store the unused 200X JC-10 (Component A) at -20 oC. Avoid repeated freeze/thaw cycles.
3. Run JC-10 assay:
3.1 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 dye-loading solution (from Step 2.2). Titration of the CCCP or FCCP may be required for optimal results with an individual cell lines.
3.2 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 dye-loading solution (see Step3.3).
3.3 Resuspend cells in 500 μL of JC-10 dye-loading solution (from Step 2.2).
3.4 Incubate the dye-loading 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.
3.5 Monitor the fluorescence intensity by using a flow cytometry in FL1 channel for the green fluorescent monomeric signal (in apoptotic cells), and the FL2 channel for the orange fluorescent 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.
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 Mitochondrial Membrane Potential Detection Kit | ||
|---|---|---|---|
| CAT# | 22206 | CAS# | N/A |
| Storage# | −20°C Sealed & desiccated & Minimized light exposure & Minimized Freezing And Thawing | Shelf Life# | 24 months |
| Ex(nm)# | N/A | Em(nm)# | N/A |
| MW# | N/A | Solvent# | N/A |
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