Mitochondrial Membrane Potential Assay Kit (JC-10)

Cat. No.: M1509036

GRADE & PURITY BioReagent

Overview

  JC-10 is an ideal fluorescent probe for detecting mitochondrial membrane potential (ΔΨm). It can rapidly and sensitively reflect changes in the membrane potential of cells, tissues, or purified mitochondria.

  When the mitochondrial membrane potential is high, JC-10 accumulates in the mitochondrial matrix and forms aggregates (J-aggregates), which emit red fluorescence. When the mitochondrial membrane potential is low, JC-10 cannot accumulate in the matrix and remains in its monomeric form, emitting green fluorescence. Thus, changes in mitochondrial membrane potential can be detected by the shift in fluorescence color. The relative ratio of red to green fluorescence is commonly used to assess the proportion of mitochondrial depolarization. A decrease in mitochondrial membrane potential is an early hallmark event of apoptosis. The transition of JC-10 fluorescence from red to green easily indicates this depolarization and serves as an early indicator of apoptosis. JC-10 monomers have maximum excitation and emission wavelengths of 490 nm and 525 nm, respectively. JC-10 aggregates (J-aggregates) have maximum excitation and emission wavelengths of 540 nm and 590 nm, respectively. The red/green fluorescence intensity ratio, measured by a fluorescence spectrophotometer, is used to determine the mitochondrial membrane potential (MMP). For observation, standard setups for red and green fluorescence are sufficient.

  The Mitochondrial Membrane Potential Assay Kit (JC-10) uses JC-10 as the fluorescent probe to rapidly and sensitively detect changes in the membrane potential of cells, tissues, or purified mitochondria. It can be used for early apoptosis detection. CCCP is included as a positive control for inducing mitochondrial membrane potential depolarization. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.

Operating Steps (for reference only)

1. Preparation of JC-10 Staining Working Solution

Take an appropriate amount of JC-10 Stain (200×). Dilute JC-10 by adding 8 mL of ddH₂O per 50 μL of JC-10 Stain (200×). Vortex vigorously to dissolve and mix the JC-10 Stain completely. Then, add 2 mL of JC-10 Buffer (5×) and mix well. This is the JC-10 Staining Working Solution. For a 6-well plate, 1 mL of working solution is needed per well.Scale accordingly for other culture vessels. For cell suspensions, use 0.5 mL of working solution per 0.5–1.0 × 10⁶ cells.

2. Setting Up a Positive Control

It is recommended to add CCCP (1 mM) to the cell culture medium at a 1:100 ratio to achieve a final concentration of 10 μM. Treat cells for 20 minutes. Then, load JC-10 as described below to detect mitochondrial membrane potential. For most cells, 10 μM CCCP treatment for 20 minutes typically causes complete loss of mitochondrial membrane potential, resulting in green fluorescence after JC-10 staining. Normal cells stained with JC-10 should show red fluorescence. For specific cell types, the optimal CCCP concentration and treatment time may vary; please refer to relevant literature.

3. For Suspension Cells

(1) Take 1×10⁵ to 6×10⁵ cells and resuspend them in 0.5 mL of cell culture medium (which may contain serum and phenol red).

(2) Add 0.5 mL of JC-10 Staining Working Solution and mix by inverting several times. Incubate at 37°C in a cell culture incubator for 20 minutes.

(3) During incubation, prepare an adequate amount of JC-10 Buffer (1×) by adding 4 mL of distilled water to every 1 mL of JC-10 Buffer (5×). Keep it on ice.

(4) After incubation, centrifuge at 4°C, 600 g for 3–4 minutes to pellet cells. Carefully aspirate the supernatant without disturbing the pellet.

(5) Wash cells twice with JC-10 Buffer (1×): Resuspend the pellet in 1 mL of JC-10 Buffer (1×), centrifuge at 4°C, 600 g for 3–4 minutes, and aspirate the supernatant. Repeat once.

(6) Resuspend the cells in a small volume of JC-10 Buffer (1×) and observe under a fluorescence microscope or confocal laser scanning microscope. Alternatively, analyze using a fluorescence spectrophotometer or flow cytometer.

4. For Adherent Cells

Note: For adherent cells intended for analysis by fluorescence spectrophotometer or flow cytometry, harvest and resuspend the cells first, then follow the protocol for suspension cells.

(1) Aspirate the medium from the 6-well plate. If necessary, wash cells once with PBS or an appropriate solution. Add 1 mL of cell culture medium (which may contain serum and phenol red).

(2) Add 1 mL of JC-10 Staining Working Solution and mix well. Incubate at 37°C in a cell culture incubator for 20 minutes.

(3) During incubation, prepare JC-10 Buffer (1×) as described in step 3.3 and keep it on ice.

(4) After incubation, aspirate the supernatant and wash the cells twice with JC-10 Buffer (1×).

(5) Add 2 mL of cell culture medium (which may contain serum and phenol red).

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

5. For Purified Mitochondria

(1) Further dilute the prepared JC-10 Staining Working Solution 5-fold with JC-10 Buffer (1×).

(2) Add 0.1 mL of purified mitochondria (total protein 10–100 μg) to 0.9 mL of the diluted JC-10 Staining Working Solution.

(3) Detection by fluorescence spectrophotometer or fluorescence microplate reader: Mix and immediately perform a time scan using a fluorescence spectrophotometer (excitation: 485 nm, emission: 590 nm). If using a microplate reader where 485 nm is unavailable, set the excitation wavelength between 475–520 nm. Alternatively, follow the wavelength settings in step 6 below.

(4) Observation by fluorescence or confocal microscope: Follow the method in step 6 below.

6. Fluorescence Observation and Analysis

For JC-10 monomers, set excitation at 490 nm and emission at 530 nm. For JC-10 aggregates (J-aggregates), set excitation at 525 nm and emission at 590 nm.

Note: It is not necessary to use the exact maximum excitation/emission wavelengths. For fluorescence microscopy, settings for JC-10 monomers can be similar to those for GFP or FITC (green fluorescence). Settings for JC-10 aggregates can be similar to those for Propidium Iodide or Cy3 (red fluorescence). Green fluorescence indicates decreased mitochondrial membrane potential, suggesting the cell is likely in early apoptosis. Red fluorescence indicates normal mitochondrial membrane potential and cell state.

Precautions

1. JC-10 Stain (200×) should be completely dissolved and mixed before use. Avoid repeated freeze-thaw cycles. JC-10 must be fully dissolved in ddH₂O before adding JC-10 Buffer (5×). Do not add JC-10 Stain to pre-prepared JC-10 Buffer (1×), as this will prevent proper dissolution and severely affect detection.

2. This kit is sufficient for 100 samples in a 6-well plate or 200 samples in a 12-well plate.

3. When washing with JC-10 Buffer (1×) after JC-10 loading, keep the buffer at approximately 4°C for better washing efficiency.

4. Complete detection within 30 minutes after JC-10 loading and washing. Keep samples on ice before detection.

5. Do not prepare all JC-10 Buffer (5×) into 1× buffer, as the 5× concentrate is needed during the procedure.

6. If precipitate forms in JC-10 Buffer (5×), ensure it is completely dissolved before use. Heating at 37°C may aid dissolution.

7. CCCP is a mitochondrial electron transport chain inhibitor and is toxic. Handle with care and appropriate protection.

8. For your safety and health, please wear a lab coat and disposable gloves during operation.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light,Room temperature,Store at -20°C

Shipped In

Ice chest + Ice pads

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.

Contents & Storage

M1509036	Component	100T	Storage
M1509036A	JC-10 Stain (200×)	100 μL×5	-20℃. Store in the dark.
M1509036B	JC-10 Buffer (5×)	80 mL	2-8℃.
M1509036C	CCCP (1mM)	200 μL	-20℃.
M1509036D	ddH₂O	90 mL	RT.