JC-1 Mitochondrial Membrane Potential Assay Kit 

ΔΨm, mitochondrial membrane potential, is an important parameter of mitochondrial function and has been used as an indicator of cell health. Variation of ΔΨm would be studied using JC-1. In healthy cells with high ΔΨm, JC-1 forms complexes known as J-aggregates. While in cells with low ΔΨm, JC-1 remains in the monomeric form. When excited at 510 nm, JC-1 monomers emit a green fluorescence with a maximum at ~527 nm. Aggregates of JC-1 emit an orange-red fluorescence with a maximum at ~590 nm.

MCE JC-1 Mitochondrial Membrane Potential Assay Kit uses JC-1, a lipophilic cationic dye, to detect the mitochondrial membrane potential in variety of cell types, as well as intact tissues and isolated mitochondria.

MCE JC-1 Mitochondrial Membrane Potential Assay Kit consists of JC-1, Phosphate-buffered saline (10×) and CCCP (50 mM in DMSO). CCCP controls should be used to confirm that the JC-1 response is sensitive to changes in membrane potential.

MCE JC-1 Mitochondrial Membrane Potential Assay Kit can be analyzed by fluorescence microscopy, flow cytometer, or a fluorescence plate reader with appropriate filter sets.

Stored at -20°C protecting from light, and is stable for up to 1 year. Avoid repetitive freeze-thaw cycles while using. For immediate use, components may be stored at 2-8°C.

1. Preparation of Phosphate-buffered saline (PBS) (1×)

a. Warm the PBS (10×) until any salt crystals are completely dissolved.

b. Dilute PBS (10×) 1:10 with ddH₂O.

2. Labeling of Cells

a. Culture cells in 6-, 12- , 24-, or 96-well plates at a density of 5-10× 10⁵ cells/mL. Incubate the cells according to your normal protocol.

b. For the control tube, allow the vial of CCCP has come to room temperature, add 1 μL of CCCP (50 mM). Incubate cells at 37℃ for 5 minutes.

c. Add 10 μL JC-1 (200 μM) per well to make the final concentration at 2 μM. Incubate cells at 37℃, 5% CO₂, for 15-20 minutes.

d. After incubation, centrifuge cells for 3-4 minutes at 400× g at 4℃, carefully aspirate the supernant.

e. Wash cells twice with PBS (1×): add 2 mL PBS (1×) to suspend cells and vortex to mix thoroughly. Centrifuge cells for 3-4 minutes at 400× g at 4℃, carefully aspirate the supernant.

f. Add 500 μL PBS (1×) to suspend cells. Analyze sample on a flow cytometer, fluorescence microscopy, or fluorescence microplate reader.

3. Additional Labeling (using Annexin V as an example, not provided in this kit)

a. After step 2.c, wash cells twice with PBS (1×): add 2 mL PBS (1×) to suspend cells and vortex to mix thoroughly. Centrifuge cells for 3-4 minutes at 400× g at 4℃, carefully aspirate the supernant.

b. Add 100 μL Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) to suspend the JC-1-stained cells.

c. Add appropriate volume of Annexin V and incubate cells at 37℃, 5% CO₂, for 15 minutes.

d. Add 400 μL Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) to suspend cells. Analyze sample on a flow cytometer, fluorescence microscopy, or fluorescence microplate reader.