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  5. 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
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100 T USD 95 In-stock

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  • Description

  • Storage

  • Protocol

  • Components

  • 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.   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.

c.   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.

d.   After incubation, centrifuge cells for 3-4 minutes at 400× g at 4°C, 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°C, 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.

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
Components JC-1 (200 μM in DMSO) Phosphate-buffered saline (10×) CCCP (50 mM in DMSO)
HY-K0601-100T 230 μL × 5 25 mL 125 μL
Documentation
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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JC-1 Mitochondrial Membrane Potential Assay Kit
Cat. No.:
HY-K0601
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