JC-1 Mitochondrial Membrane Potential Assay Kit

Cat. No.: HY-K0601: FAQs
Manual SDS

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

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100 T	USD 95	In-stock	Estimated Time of Arrival: December 31

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Description
Storage
Protocol
Documentation

Description
& Advantages

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.

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Storage

Stored at -20°C protecting from light, and is stable for up to 12 months. Avoid repetitive freeze-thaw cycles while using. For immediate use, components may be stored at 2-8°C.

Protocol

1. Labeling of Cells

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

b. Ensure that the JC-1 and DMSO has equilibrated to room temperature, and then prepare a 200 μM stock solution by dissolving the contents of one vial in 230 μL of the DMSO provided.

c. 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°C for 5 minutes.

d. Add 10 μL JC-1 (200 μM) per well to make the final concentration at 2 μM. Incubate cells at 37°C, 5% CO₂, for 15-20 minutes. If additional labeling followed, for example with an annexin V, begin with step 2.a. If not, proceed with step 1.e.

e. After incubation, centrifuge cells for 3-4 minutes at 400× g at 4°C, carefully aspirate the supernant.

f. 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°C, carefully aspirate the supernant.

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

2. Additional Labeling with Annexin V

a. After step 1.d, 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°C, 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 5 μL Annexin V and incubate cells at 37°C, 5% CO₂, for 15 minutes.

Note: 5 μL is appropriate for Annexin V, usage dose may vary according to different suppliers.

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.

Components	JC-1 (200 μL in DMSO)	Phosphate-buffered saline (10×)	CCCP (50 mM in DMSO)
HY-K0601-100T	230 μL × 5	25 mL	125 μL

Documentation

Manual (188 KB) SDS (874 KB)

Help & FAQs

Do most proteins show cross-species activity?
Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

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