Mitochondrial membrane potential detection kit (JC-1)

1 、 JC-1 Preparation of dyeing working solution
Six-well plate required per well JC-1 The amount of dyeing working solution is 1mL , of other culture vessels JC-1 The amount of staining working solution used is similar: for the cell suspension per 50 ～ 100 Ten thousand cell needs 0.5mLJC-1 Dyeing Working Fluid. Take appropriate amount JC-1 （ 200× ), according to each 50μLJC-1 （ 200× ) join 8mL Proportional dilution of ultrapure water JC-1 。 Violently shake to fully dissolve and mix well JC-1 。 Then add 2mLJC-1 Staining buffer ( 5× ), after mixing, it is JC-1 Dyeing Working Fluid.

2 Settings of Positive Control:
Put the kit provided CCCP （ 10mM ) Recommended as per 1∶1000 The proportion is added to the cell culture solution, and diluted to 10μM , treating cells 20 Minutes. Subsequently loading as follows JC-1 , the mitochondrial membrane potential was detected. For most cells, typically 10μMCCCP dispose 20 After minutes, the membrane potential of mitochondria will be completely lost, JC-1 After staining, it should show green fluorescence; And normal cells through JC-1 Red fluorescence should be shown after staining. For specific cells, CCCP The concentration and duration of action may vary, so you need to refer to relevant literature to decide.

3 For suspension cells,
Take 10 ～ 60 Thousand cells, resuspended in 0.5mL In the cell culture medium, the cell culture medium may contain serum and phenol red.
join 0.5mLJC-1 Dye the working solution, reverse it several times and mix well. In cell incubator 37℃ incubation 20 Minutes.
During the incubation, according to each 1mLJC-1 Staining buffer ( 5× ) join 4mL Proportion of distilled water, prepare an appropriate amount JC-1 Staining buffer ( 1× ) and placed in an ice bath.
37℃ After the incubation is completed, 600g4℃ Centrifugation 3 ～ 4 Min, the cells were pelleted. Discard the supernatant and be careful not to aspirate the cells as much as possible.
use JC-1 Staining buffer ( 1× ) washing 2 Time: Join 1mLJC-1 Staining buffer ( 1× ) resuspending cells, 600g4℃ Centrifugation 3 ～ 4 Minutes, the cells were pelleted and the supernatant was discarded.
Re-join 1mLJC-1 Staining buffer ( 1× ) resuspending cells, 600g4℃ Centrifugation 3 ～ 4 Minutes, the cells were pelleted and the supernatant was discarded. Use an appropriate amount JC-1 Staining buffer ( 1× ) After resuspension, observe with a fluorescence microscope or laser confocal microscope, or detect with a fluorescence spectrophotometer or analyze with a flow cytometer.

4 For adherent cells,
Note: For adherent cells, if you want to use fluorescence spectrophotometer or flow cytometer detection, you can collect the cells first, and refer to the detection method of suspended cells after resuspension.
For one well of a six-well plate, the culture medium is aspirated and, if necessary, can be used according to the specific experiment PBS Or other suitable solution wash that cells once and add 1mL Cell culture fluid. The cell culture medium may contain serum and phenol red. join 1mLJC-1 Dyeing working solution, mix thoroughly. In cell incubator 37℃ incubation 20 Minutes.
During the incubation, according to each 1mLJC-1 Staining buffer ( 5× ) join 4mL Proportion of distilled water, prepare an appropriate amount JC-1 Staining buffer ( 1× ) and placed in an ice bath.
37℃ After the incubation, the supernatant was aspirated and treated with JC-1 Staining buffer ( 1× ) washing 2 Times.
join 2mL The cell culture medium may contain serum and phenol red.
Observe under fluorescence microscope or laser confocal microscope.

5 For purified mitochondria
Put the prepared JC-1 Reuse of dyeing working solution JC-1 Staining buffer ( 1× ) dilution 5 Times.
0.9mL5 Double diluted JC-1 Add to the dyeing working solution 0.1mL The total protein content was 10 ～ 100μg Purified mitochondria. Detection with a fluorescence spectrophotometer or a fluorescence microplate reader: After mixing, time scan with a fluorescence spectrophotometer directly ( timescan ), the excitation wavelength is 485nm , emission wavelength is 590nm 。 If a fluorescent microplate reader is used, the excitation wavelength cannot be set to 485nm When, you can 475 ～ 520nm The excitation wavelength is set within the range. In addition, you can also refer to the following steps 6 The wavelength setting in is performed for fluorescence detection.
Observation by fluorescence microscope or laser confocal microscope: the method is the same as the following steps 6 。

6 , fluorescence observation and result analysis detection
JC-1 The excitation light can be set to 490nm , the emitted light is set to 530nm ； Detection JC-1 Polymer, the excitation light may be set to 525nm , the emitted light is set to 590nm 。 Note that it is not necessary to set the excitation light and the emission light at the maximum excitation wavelength and the maximum emission wavelength when measuring fluorescence here. If observed using a fluorescence microscope, detect JC-1 When monomer, you can refer to the settings when observing other green fluorescence, such as observing GFP Or FITC Time setting; Detection JC-1 Polymer may be observed with reference to other red fluorescence such as propidium iodide or Cy3 When setting. The appearance of green fluorescence indicates that the mitochondrial membrane potential decreases, and the cell is probably in the early stage of apoptosis. The appearance of red fluorescence indicates that the mitochondrial membrane potential is relatively normal and the state of cells is also relatively normal.

Store at 4 ℃, protected from light, shelf life for one year