Mitochondrial Membrane Potential Assay Kit (with JC-1) (E-CK-A301)

Q1:Why is the resulting graph JC-1 slanted?

The monomers and polymers of JC-1 exist in both normal and apoptotic cells. In the process of cell apoptosis, JC-1 slowly dissociates from polymer to monomer, and the ratio of polymer to monomer changes. Therefore, when drawing the door, it is generally gathered in the third quadrant, and when drawing the door to distinguish between normal cells and apoptotic cells, it needs to be divided in the third quadrant.

Q2:What are the precautions of JC-1 imaging method?

① The microscope parameters are consistent; ② Dissolve the buffer and observe whether there are small red particles. If the buffer does not dissolve, centrifuge at 12000rpm and take the supernatant. (3) Apoptotic cells stick weakly to the flask and will float in the medium. If fluorescence microscopy is used, the results are relatively inaccurate, and flow cytometry is recommended for detection. Before collecting cells, take a photo at 100x magnification under normal white light (200x for small cells, such as lymphocytes) to check morphological changes.

Q3:Is JC-1 kit able to detect tissue samples or tissue section samples?

It is not recommended to do so. Cells may suffer apoptosis or damage due to mechanical manipulation during preparation into cell suspension, in which case the results of JC-1 may be inaccurate. In addition, this kit can only detect live single-cell suspensions and cannot be used to slice samples.

Q4:Is it able to use E-CK-A301 for cells with GFP?

JC-1 needs to detect the changes of fluorescence signal in both Ex/Em = 514/529 and Ex/Em = 585/590 at the same time to determine the changes of mitochondrial membrane potential, and there is fluorescence interference with GFP, so this kit cannot be applied to cells with GFP.

Q5:Is it able to measure red fluorescence only when operating JC-1 assay?

JC-1 exists in monomer and polymer form, and their emission spectra are different. In normal cells with high mitochondrial membrane potentia, JC-1 accumulates in the matrix of mitochondria as polymer, which producing red fluorescence. Since the decreasing of mitochondrial membrane potential during early stage of apoptosis, JC-1 can no longer be accumulated in the mitochondrial matrix, that becomes monomer with green fluorescence. Therefore, both red and green fluorescence need to be observed.

Q6:Can tissue samples for JC-1 and ROS be streamed if they're frozen in dry ice? Is there another way?

After the tissue sample is dry frozen, basically the cell morphology may have been destroyed and can not be used for flow detection. You can look at ELISA or metabolic indicators related to research, such as apoptosis of Bax, Caspase or oxidative stress of SOD, MDA and so on.

Q7:Can the mitochondria be extracted to test JC-1?

It's theoretically possible, but we haven't tested it yet. We cannot determine the amount of JC-1 and the amount of extracted mitochondria, and may need to explore optimization.

Q8:Can the JC-1 mitochondrial assay kit be used for plant tissue samples?

Plant tissues have cell walls and chloroplasts, which may interfere with JC-1 detection. We have not validated this application and cannot confirm its effectiveness. If testing, we recommend consulting relevant literature and conducting preliminary experiments.

Q9:Can Mito-Tracker Green be replaced by JC-1?​

No. Mito-Tracker Green is a green fluorescent probe specifically designed for labeling mitochondria in live cells. Unlike JC-1, its staining mechanism is independent of mitochondrial membrane potential.

Q10:Can JC-1 be used to detect mitochondrial membrane potential in heart tissue samples?

We do not recommend using tissue samples to prepare single-cell suspension for JC-1, because the operation of preparing single-cell suspension has a great impact on cells and will directly affect the experimental results of JC-1.