JC-1 Mitochondrial Membrane Potential Assay Kit

 

product name	Identification of product	model
JC-1 Mitochondrial Membrane Potential Assay Kit	G1515-100T	100T

 

 

JC-1 is a cationic carbonyl cyanine dye, which can pass through the cell membrane and aggregate towards mitochondria under the action of mitochondrial membrane potential. It is an ideal fluorescent probe widely used in detecting mitochondrial membrane potential. When the mitochondrial membrane potential is high, the dye aggregates in the matrix of mitochondria, showing red fluorescence(Ex=585 nm,Em=590 nm);When the mitochondrial membrane potential is low, JC-1 is predominantly a monomer exist in cytoplasm, and showing green fluorescence(Ex=514 nm,Em=529 nm).Therefore, the change of mitochondrial membrane potential can be judged according to the transformation of fluorescence color and the change of proportion, and the decrease of mitochondrial membrane potential is also an important marker of early apoptosis.

JC-1 Mitochondrial Membrane Potential Assay Kit, is based on JC-1 fluorescent probe, which has improved easy precipitation problem and other shortcomings. This kit can be used to quickly and sensitively detect mitochondrial membrane potential changes in cell or purified mitochondria for determining the early apoptosis, and is also a common method used to detect early cell apoptosis. A total of 100 6-well plate samples can be tested in this kit.

In addition, this kit also provides CCCP (Carbonyl cyanide 3-chlorophenylhydrazone) reagent, a proton carrier (hydrogen ionophore) and decoupler of oxidative phosphorylation in mitochondria, which can promote changes in the permeability of mitochondria to hydrogen ions, resulting in the decrease or loss of mitochondrial membrane potential, which can be used as a positive control for inducing the decrease of mitochondrial membrane potential.

 

 

JC-1 dye(500×)should be stored at -20℃,desiccated and protected from light, and avoid repeated freezing and thawing;

JC-1 buffer and JC-1 diluent may be stored at 4℃. Valid for 12 months.

 

 

Component Number	Component	G1515-100T
G1515-1	JC-1 dye(500 ×)	4 x 50 μL
G1515-2	JC-1 buffer (10 x)	50 mL
G1515-3	JC-1 diluent	100 mL
G1515-4	CCCP(100 mM)	50 μL
Manual		1 pc

 

 

1. prepare the working solution of JC-1 dye(2x) and JC-1 buffer(1x):

a. Take 2 μL of JC-1 solution (500×) and 900 μL of JC-1 dilution solution and mix well, then add 100 μL of JC-1 buffer (10×) and mix well by vortexing and shaking to prepare JC-1 working solution for spare use (take care to prepare this solution in order);

b. Take an appropriate amount of JC-1 buffer (10×) diluted with deionized water to formulate 1×JC-1 buffer and set aside (1×JC-1 buffer is used for subsequent steps, such as washing, if not otherwise specified).

2. prepare a positive control(optional ):

a. Take an appropriate amount of CCCP (100 mM) and dilute 1000 times with cell culture medium to obtain 100 μM CCCP working solution;

b. Incubate the cells with CCCP working solution for about 30 minutes, and then follow the JC-1 staining procedure below to detect the mitochondrial membrane potential.

Note: For most of the cells, after treatment with the above treatments, compared with the normal group, JC-1 can be seen to exist mostly in the cytoplasm as monomers after induction by CCCP treatment, showing brighter green fluorescence and weaker red fluorescence; However, for some cells, the concentration and incubation time of CCCP may be different, please refer to reference or do experiment to find the optimal conditions.

3. Cell stain and analyze

a. Take 1-6×105 cells, centrifuge at 1000 g for 3-5 min to remove the original medium, add JC-1 buffer and wash once, then centrifuge to remove JC-1 buffer and resuspend with 500 μL of cell culture medium (serum and phenol red can be included).;

b. Then add 500 μL of JC-1 working solution and incubate in a CO2 incubator for 15-30 min (generally 20 min is sufficient, or the incubation time can be adjusted according to the situation), protected from light;

c. At the end of the incubation, the cells were still treated as conventional suspension cells, and after centrifugation to remove the JC-1 working solution, the cells were washed twice with JC-1 buffer ;

d. After resuspension in appropriate amount of JC-1 buffer (1×) or cell culture solution (phenol red free is recommended), it was observed by fluorescence microscope or laser confocal microscope, or analyzed by flow cytometry and other instruments..

4. Adherent cell staining operation (6-well plate as an example):

For adherent cells, if they need to be detected by flow cytometry, the cells can be collected first, resuspended and then referred to the assay for suspension cells.

a. After removing the original cell culture medium containing drugs or other treatments, add 1 mL of JC-1 buffer and wash 1-2 times;

b. Add 1 mL of cell culture medium (may contain serum and phenol red);

c. Add 1 mL of JC-1 working solution, shake gently to mix well, and incubate for 15-30 min in a CO2 incubator protected from light;

d. At the end of incubation, the supernatant was aspirated, and with JC-1 buffer, washed twice;

e. Add 2 mL of JC-1 buffer (1×) or cell culture solution (phenol red free recommended);

f. Observed under a fluorescence microscope or laser confocal microscope or analyzed by an instrument such as a flow cytometer.

5. For the extraction of purified mitochondria

a. 900 μL of JC-1 working solution was supplemented with 100 μL of total protein amounting to 10-100 μg of purified mitochondria;

b. After mixing, it was scanned and detected using an instrument such as a fluorescence spectrophotometer (with an excitation wavelength of 485 nm and an emission wavelength of 590 nm) or an enzyme labeling instrument (when the excitation wavelength could not be set to 485 nm, the excitation wavelength could be set within the range of 475-520 nm), or it could be observed by fluorescence or laser confocal microscopy, which could be detected with reference to the wavelength setting in step 6.

6. Fluorescence detection and analysis

a. The wavelength parameters for detecting JC-1 monomer are Ex=490 nm, Em=525 nm; the wavelength parameters for JC-1 polymer are Ex=525 nm, Em=590 nm; note that it is not necessary to set the excitation and emission at the maximum excitation and emission wavelengths in the determination of fluorescence here, and that the parameter settings can be adjusted according to the situation around this parameter;

b. When taking pictures with fluorescence microscope, it is recommended to use the normal group as a standard to determine the exposure time for red and green fluorescence, so that the red fluorescence effect of the normal group is clear and the green fluorescence effect is darker, and to take pictures of the fluorescence of the processing group with the exposure time for red and green fluorescence of the normal group, respectively;

4. Judgment of the results: the cell state is normal, mainly showing red fluorescence, indicating that its mitochondrial membrane potential is normal; if there is a significant increase in green fluorescence, indicating that the mitochondrial membrane potential has declined, which may be in the early stage of apoptosis.

 

 

1. In order to fully dissolve JC-1, follow the sequence of operation for JC-1 working solution preparation.

2. It is recommended to test the samples within 30 min after JC-1 incubation.

3. JC-1 solution, JC-1 buffer (10×) can be dissolved in an appropriate warm bath if precipitation is produced.

4. For JC-1 final assay, it is recommended to use phenol red free cell culture solution to avoid background color interference, and phenol red at the incubation stage of JC-1 staining has no effect on the results.

5. If less JC-1 solution is used at a time, which may involve multiple repeated freezing and thawing, please dispense as appropriate and try to avoid multiple repeated freezing and thawing of JC-1 solution.

6. Please wear a lab coat and disposable gloves in operation.

 

For Research Use Only!