How to carry out mitochondrial membrane potential detection(JC-1)?

Mitochondrion is an organelle which is coated by two-ply cell membrane and and lives in most of cells. It creates energy in cells and also it is the major place for cells to do aerobic respiration, so it is also called the power house of cells. Mitochondrion also participates in the process of cell apoptosis, cell differentiation and cell signaling, having the capability to regulate cell growth and cell cycle.

Many researches showed that mitochondrion is closely related with cell apoptosis, in which JC-1 –  mitochondrial membrane potential was thought as one of the earliest process happened in cell apoptosis. It happens before showing the features like chromatin condensation and DNA fracture.

Once the mitochondrial membrane potential happens, then cell apoptosis will be irreversible.

 

Figure 1: Graphical abstract

(Picture source: https://www.mdpi.com/1422-0067/19/7/1985)

Required instruments or reagents for mitochondrial membrane potential detection:

  1. Flow cytometeror Fluorescence microscope
  2. High-speed centrifugeand CO2 incubator
  3. Micropipetteand 1.5ml Microtube
  4. Glass slideand Coverslip
  5. PBS and Sterilized pure water

Attention points before carrying out mitochondrial membrane potential detection

  1. Centrifuge before using reagent
  2. JC-1 Stainshall be kept away from light
  3. The quantity of cultured cells shall not greater than 1×106
  4. Set one positive control and one negative control group
  5. Extract mitochondrion before detection

Below three products are recommended to carry out mitochondrial membrane potential detection:

CAT# Product Name Kit Components Size&Price
KTP4004 ExKine™ Mitochondrion Extraction Kit (Tissue)

 

• Lysis Buffer A (5×)

• Lysis Buffer B (5×)

• Storage Buffer

$40/50T

$120/200T

 

KTP4003 ExKine™ Mitochondrion Extraction Kit (Cultured Cells)

 

• Lysis Buffer A (5×)

• Lysis Buffer B

• Lysis Buffer C

• Storage Buffer

$40/50T

$120/200T

KTA4001 Mitochondrial Membrane Potential Assay Kit (JC-1)

 

• JC-1 Stain

• CCCP (10 mM)

• Assay Buffer (5×)

$40/20T

$120/100T

$360/500T

Especially Abbkine Mitochondrial Membrane Potential Assay Kit (JC-1)(CAT#KTA4001) was optimized using Hela cells, and please refer to the proposed protocol as below:

  1. Quantification by Flow Cytometry
  2. Treat cells with the desired method, and set up parallel control experiments.

For Negative Control: Treat cells with vehicle only.

For Positive Control: Treat cells with CCCP at 5-20 Min a 37oC, 5% CO2 incubator for 20

to 30minutes.

  1. For non-adherent cells, Collect 1×106cells by centrifugation (4oC, 300g, 5min). Wash with ice-cold PBS twice and discard the PBS. For adherent cells, using Trypsin (EDTA free) to digest cells firstly and then centrifugation.
  2. Resuspend the cells pellet in 500uL Staining Solution.
  3. Incubate the cells at 37°C for 15-30 minutes in the dark.
  4. Centrifuge cells at 500 g and discard supernatant.
  5. Wash cell pellet with PBS and repeat step 5.
  6. Resuspend cell pellet in 1 ml of the pre-warmed PBS and analyze cells immediately byflow cytometry.
  7. Detection by Fluorescence Microscopy
  8. For non-adherent cells: Follow the protocol for flow cytometry from step 1 to step 6 and

place the cell suspension from Step A.6 on a glass slide. Cover the cells with a glass

coverslip. Analyze cells by fluorescence microscopy using the appropriate filters as soon

as possible.

  1. For adherent cells: the suggested protocol is as below:

2.1. Grow cells directly on a coverslip in 24 well dish. Incubate in a CO2 Incubator at 37°C

for at least 24 hours before treatment.

2.2. Treat cells with the desired method and Prepare a positive control as mentioned in

Step A.1.

2.3. Wash cells with PBS twice and discard the PBS.

2.4. Add 0.5 mL of Staining solution to cells and incubate at 37°C for 15-30 minutes in the

dark.

2.5. Discard the supernatant and wash cells with PBS twice.

2.6. Overlay the cells with the pre-warm growth medium and observe the cells under a

fluorescence microscope

Figure 2: Hela cells stained with Abbkine Mitochondrial Membrane Potential Assay Kit (JC-1). A: Red fluorescence indicates healthy mitochondria, B: Green fluorescence indicates mitochondria in poor health (30min incubation in 20uM CCCP).

Abbkine provides you cost effective price on one stop reagent solution to carry out mitochondrial membrane potential detection(JC-1). Feel free to contact service@abbkine.com, based on above price, we can extra apply academic or distributor discount for you in collaboration!

About Abbkine Scientific Co., Ltd.

Abbkine offers services for global scientists in the fields of proteomics and cytology, and is committed to the innovation and development of various scientific reagents related to proteomics and cytology, to promote the development of fields including life science research and drug discovery. Proteomics products cover the preparation of samples (protein extraction, purification, coupling), protein quantification, antibodies and kits for protein detection. Cytology products involve cytokines (cell culture), cell status detection, cell staining, organelle extraction, cell metabolism and cytopathology reagents (kits). Abbkine relies on the product portfolio and unique marketing support as the main market strategy and product innovation mode, to help your research career!

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