JC-1 Mitochondrial Membrane Potential Detection Kit

Product Introduction

JC-1Mitochondrial membrane potential detection kit（JC-1 Mitochondrial Membrane Potential Assay Kit）It is a kind ofJC-1A fluorescent probe kit for rapid and sensitive detection of changes in cell, tissue, or purified mitochondrial membrane potential. The decrease in mitochondrial membrane potential is a hallmark event in the early stage of cell apoptosis. Therefore, this kit can be used for early detection of cell apoptosis.

JC-1It is a widely used method for detecting mitochondrial membrane potential△ΨmIdeal fluorescent probe for positioning.JC-1Dyes accumulate in mitochondria in a potential dependent manner and can be used to detect the membrane potential of cells, tissues, or purified mitochondria. In normal mitochondria,JC-1Aggregate in the mitochondrial matrix to form polymers, which emit strong red fluorescence（Ex=585 nm, Em=590 nm）； Unhealthy mitochondria, due to a decrease or loss of membrane potential,JC-1Can only exist in the cytoplasm in the form of monomers, producing green fluorescence（Ex=514 nm, Em=529 nm）.JC-1Not only can it be used for qualitative detection, but the change in color can directly reflect the change in mitochondrial membrane potential. It can also be used for quantitative detection, as the degree of depolarization of mitochondria can be determined by red/Measured by the ratio of green fluorescence intensity.

Product Composition

saveAnd transportation：-20Store in a dark and dry place at ℃, at least1Valid annually. Transportation:ice packtransportation

Usage JC-1 Mitochondrial Membrane Potential Detection Kit

1. JC-1Preparation of dyeing working solution

For six well plates, this kit can detect100A sample. Required for each holeJC-1The amount of dyeing working solution is1mlOther cultivation vessels JC-1The dosage of dyeing working solution is the same; For each cell suspension 50～100Ten thousand cells are needed 0.5mL JC-1Dyeing working fluid. Take an appropriate amount JC-1（200×）According to each50μL JC-1（200×）Join 8mLProportional dilution of ultrapure water JC-1Thoroughly shake and dissolve thoroughly, then mix well JC-1Then join in 2mL JC-1Staining buffer solution（5×）After mixing, it is JC-1Dyeing working fluid.

[Attention]:We must first JC-1（200×）Dissolve and mix thoroughly with ultrapure water (provided in the reagent kit) before adding JC-1Staining buffer solution（5×）. Cannot be prepared first JC-1Staining buffer solution（1×）Join again JC-1（200×）This way JC-1It will be difficult to fully dissolve and will seriously affect subsequent testing.

2. Setting of positive control

CCCP（10mM）Recommended Press 1∶1000 Add the proportion to the cell culture medium and dilute to 10μMProcessing cells 20minThen load according to the following method JC-1Perform detection of mitochondrial membrane potential. For most cells, typically 10μM CCCPhandle 20 minThe membrane potential of mitochondria willwanquanLoss,JC-1After staining, it should exhibit green fluorescence upon observation; And normal cells JC-1After staining, it should display red fluorescence. For specific cells,CCCPPossible concentration and duration of action

Different, please refer to relevant literature and materials to make your own decision.

3. For suspended cells

1）取10～60 Ten thousand cells, suspended again 0.5mLCell culture medium can contain serum and phenol red.

2）Join0.5mL JC-1Dyeing solution, invert several times and mix well. In a cell culture incubator 37Incubation at ℃20min.

3）During the incubation period, according to the 1mL JC-1Staining buffer solution（5×）Join 4mLThe proportion of distilled water, prepare an appropriate amount JC-1Staining buffer solution（1×）And place it in an ice bath.

4）37After incubation at ℃,600×g4℃ centrifugation3～4minSettling cells. Discard the supernatant and be careful not to aspirate cells as much as possible.

5）Use JC-1Staining buffer solution（1×）Washing 2 Next: Join 1mL JC-1Staining buffer solution（1×）Resuspended cells,600×g4℃ centrifugation3～4minSettle the cells and discard the supernatant. add in 1mL JC-1Staining buffer solution（1×）Resuspended cells,600×g4℃ centrifugation3～4minSettle the cells and discard the supernatant.

6）Use an appropriate amount again JC-1Staining buffer solution（1×）After resuspension, observe with a fluorescence microscope or laser confocal microscope, or use fluorescence spectroscopy Detection by photometer or analysis by flow cytometry.

4. For adherent cells

[Attention]For adherent cells, if you want to use a fluorescence spectrophotometer or flow cytometer for detection, you can first collect the cells, resuspend them, and then refer to the suspended cell method for detection.

1）For one hole of the six well plate, remove the culture medium and use it if necessary based on specific experiments PBSWash the cells once with another appropriate solution and add 1mLCell culture medium. Cell culture medium can contain serum and phenol red.

2）Join 1mL JC-1Staining solution, mix thoroughly. In a cell culture incubator 37Incubation at ℃20min.

3）During the incubation period, according to the 1mL JC-1Staining buffer solution（5×）Join 4mLThe proportion of distilled water, prepare an appropriate amount JC-1Staining buffer solution（1×）And place it in an ice bath.

4）37After incubation at ℃, remove the supernatant and useJC-1Staining buffer solution（1×）Washing 2 Next time.

5）Join 2mLCell culture medium, which may contain serum and phenol red.

6）Observe under a fluorescence microscope or laser confocal microscope.

5. For purified mitochondria

1）Prepare the prepared JC-1Reuse of dyeing working solution JC-1Staining buffer solution（1×）Dilution 5 Twice.

2）0.9mL 5Diluted multiple times JC-1Add to the dyeing working solution 0.1mLThe total protein content is 10～100μgPurified mitochondria.

3）Detection using a fluorescence spectrophotometer or fluorescence enzyme-linked immunosorbent assay (ELISA): After mixing, directly use a fluorescence spectrophotometer for time scanning（time scan）The excitation wavelength is485nmThe emission wavelength is 590nmIf using a fluorescent enzyme-linked immunosorbent assay (ELISA) reader, the excitation wavelength cannot be set to 485nmAt that time, it is possible to475～520nmSet the excitation wavelength within the range. Additionally, you can also refer to the following steps 6 Set the wavelength for fluorescence detection.

4）Observe with fluorescence microscope or laser confocal microscope: the method is the same as the following steps 6.

6. Fluorescence observation and result analysis

detection JC-1When using monomers, the excitation light can be set to 490nmSet the emission light to 530nm; Testing JC-1When using polymers, the excitation light can be set to 525nmSet the emission light to 590nm.

[Note]1】When measuring fluorescence here, it is not necessary to set the excitation light and emission light at the maximum excitation wavelength and maximum emission wavelength. If observed and detected using a fluorescence microscope JC-1Polymer can be used to detect propidium iodide or Cy3The standard bandpass filter used. detection JC-1Monomers can be detected using conventional methods FITCor GFPThe standard bandpass filter. The appearance of green fluorescence indicates a decrease in mitochondrial membrane potential, and the cell is likely in the early stage of apoptosis. The appearance of red fluorescence indicates that the mitochondrial membrane potential is relatively normal and the state of the cell is also relatively normal.

Precautions

1）JC-1（200×）In4At lower temperatures such as ℃ and ice bath, it will solidify and stick to the bottom, wall or cover of the centrifuge tube20～25Incubate in a water bath at ℃ for a moment until completely melted before use.

2）Finished loading JC-1Later use JC-1Staining buffer solution（1×）When washing, make JC-1Staining buffer solution（1×）Maintain 4Around ℃, the washing effect is better at this time.

3）JC-1After loading and washing the probe, try to use it as much as possible 30minComplete subsequent testing internally. Ice bath storage is required before testing.

4）Please do notJC-1Staining buffer solution（5×）All prepared intoJC-1Staining buffer solution（1×），This reagent kit needs to be used directly during useJC-1Staining buffer solution（5×）.

5）If discovered JC-1Staining buffer solution（5×）There is sediment in it, which must be completely dissolved before use. It can be used in 37Heating at ℃ promotes dissolution.

6）CCCPAs a mitochondrial electron transport chain inhibitor, it is toxic. Please be careful with protection.

7）For your safety and health, please wear lab coats and disposable gloves when operating.

Related Products

JC-1JC-1 Partial ReferencesJC-1 Mitochondrial Membrane Potential Detection Kit

(1) [IF=9.7]Contribution of ferroptosis and GPX4’s dual functions to osteoarthritis progression-EBioMedicine Published:January 27, 2022 DOI: https://doi.org/10.1016/j.ebiom.2022.103847

The original data for this figure comes from reference (1).

(2) [IF=11.4]VDR regulates mitochondrial function as a protective mechanism against renal tubular cell injury in diabetic rats. -Redox Biology -Volume 70, April 2024, 103062.V

The original data for this figure comes from reference (2).

(3)[IF=11.1]LONP1 targets HMGCS2 to protect mitochondrial function and attenuate chronic kidney disease-EMBO Mol Med(2023)15: e16581https://doi.org/10.15252/emmm.202216581

Immunofluorescence co localization analysis of LONP1, HMGCS2, and mitochondria in mPTC (top, scale: 20um) and human kidney tissue (bottom, scale: 200um).

The original data for this figure comes from reference (3).