Detection of mitochondrial membrane potential

2019-07-18Author:adminpraise:1

Detection principle

The decrease of mitochondrial membrane potential is a marker event in the early stage of apoptosis. It occurs before the appearance of nuclear apoptotic characteristics (chromatin concentration and DNA fragmentation). Once the mitochondrial membrane potential collapses, apoptosis is irreversible.

In normal cells, the mitochondrial membrane potential is high, JC-1 gathers in the matrix of mitochondria to form a polymer, which yields a red to orange colored emission (590±17.5 nm). In the early stage of apoptosis, the mitochondrial membrane potential decreases, JC-1 can’t gather and it is a monomer which yields green fluorescence with emission of 530±15 nm.

Analysis by the Flow cytometry, normal cells are double positive with red and green fluorescence and the apoptosis cells are single positive with only green fluorescence.

Experimental Procedure:

  1. Deploy the 1×JC-1 Assay Buffer and JC-1 Staining Buffer according to the requirement of the experiment. See the instructions above for detail. The buffer should be stored at 4°C.

  2. Set the positive Control. See the instructions above for detail. Induce apoptosis of suspension cells with reagents of interest.

  3. Collect the cells, centrifuge at 300 g for 5 min, and discard the supernatant. Add appropriate amount of PBS to resuspend gently and count the cells.

TipsCell viability is the key to the experiment. When the adherent cells are used for apoptotic detection, treatments like digestion may increase the ratio of necrotic or apoptotic cells and cause uncontrollable effects on the experimental results. Please be aware

  1. Split the cell suspension into tubes, 1-5 × 105 cells for each. Centrifuge at 300 g for 5 min, discard the supernatant. Add PBS to wash the cells and discard the supernatant.

  2. Add 500 μL JC-1Staining Buffer to resuspend the cells. Incubate at 37°C, 5% CO2 incubator for 15-20 min.

TIP: The incubation time is depending on cell types. In general, mammalian cells are recommended at 37°C. Other species should according to cell culture conditions.

  1. Centrifuge at 300 g for 5 min, discard the supernatant. Add 500 μL pre-cold 1x JC-1 Assay Buffer to wash the cells twice.

  2. Add 500 μL pre-cold 1x JC-1 Assay Buffer to resuspend the cells.

  3. Analyze the cells immediately with proper machine settings. Otherwise, place the cells on ice in the dark and analyze within 0.5 h.

Detection by microscopy or flow cytometry

   A. Analysis by Fluorescence Microscopic Observation

     1. Drop the cell suspension on a slide, cover the slide, and observe with a fluorescence microscope.

     2. For adherent cells, slides can also be used to culture cells and induce apoptosis directly, and wash cells twice with PBS.

       a)Add 100 μ L 1x JC-1 Staining Buffer to the slide.

       b)Incubate in an incubator with 37oC, 5% CO2 for 15-20 min, wash 1-2 times with 1 x JC-1 Staining Buffer

       c)Observe with the fluorescence microscope.

When detecting JC-1 monomer, the excitation light can be set to 490 nm and the emission light to 530 nm.

When detecting the JC-1, the excitation light can be set to 525 nm and the emission light to 590 nm.

Tips:

It is not necessary to set the excitation and emission light at the maximum wavelengths for fluorescence determination here.

If observed by fluorescence microscopy, JC-1 monomer can be detected with reference to other settings when observing green fluorescence, such as GFP or FITC.

Detection of JC-1 polymer can refer to other red fluorescence, such as PI.

The presence of green fluorescence indicated that mitochondrial membrane potential decreased, and the cell is probably in the early stage of apoptosis. The appearance of red fluorescence indicated that mitochondrial membrane potential and cell status are normal.

B. Flow cytometry analysis

Flow cytometry can be used to detect apoptosis (Ex = 488 nm; Em = 530 nm).

Green fluorescence is usually detected by FL1 in FITC channel and red fluorescence by FL2 in PI channel.

Normal cells {FL-1 bright, FL-2 bright; R1}, apoptotic cells {FL-1 bright, FL-2 dark; R2}, the location of the gate is different with the type of root starch cells, experimental conditions and so on vary.

Negative control (untreated normal cells) and positive control (treated with CCCP) must be set during the experiment. The location of the gate is determined by two-parameter scatter plots of the negative and positive control groups.

Apply for
*Product Name:
*Catalog Number:
*Name:
*Email:
*Message:
*Country:
How do you know
Elabscience:

*Captcha: