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  2. JC-1 Mitochondrial Membrane Potential Assay Kit

JC-1 Mitochondrial Membrane Potential Assay Kit 

Cat. No.: HY-K0601
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.

JC-1 Mitochondrial Membrane Potential Assay Kit
Size Price Stock Quantity
Free Sample (10 T)   Apply Now  
100 T USD 240 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 490 nm, JC-1 monomers emit a green fluorescence with a maximum at ~520 nm. Aggregates of JC-1 emit an orange-red fluorescence with a maximum at ~590 nm.

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.

Documentation

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Product Name:
JC-1 Mitochondrial Membrane Potential Assay Kit
Cat. No.:
HY-K0601
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