Mitochondrial Membrane Potential Assay Kit (JC-1), IC-6568

Product Introduction

This product is a kit that uses JC-1 as a fluorescent probe to detect changes in mitochondrial membrane potential in cells, tissues, or purified mitochondria, and can be used to detect the early stages of apoptosis. JC-1 is a widely used fluorescent probe for detecting mitochondrial membrane potential (¦¤¦·m). Loss of mitochondrial membrane potential is considered an early event in apoptosis. Mitochondrial uptake of JC-1 can effectively distinguish apoptotic cells from normal cells. When mitochondrial membrane potential is high, JC-1 exists as J-aggregates in the mitochondrial matrix and emits red fluorescence; when mitochondrial membrane potential is low, JC-1 cannot accumulate in mitochondria and instead exists as monomers dispersed throughout the cell, emitting green fluorescence. Therefore, the shift in fluorescence color reflects changes in mitochondrial membrane potential. The ratio of red to green fluorescence is commonly used to measure the extent of mitochondrial depolarization. JC-1 monomer has maximum excitation at 514 nm and maximum emission at 529 nm; JC-1 J-aggregates have maximum excitation at 585 nm and maximum emission at 590 nm. The kit provides CCCP (10 mM) as a positive control for inducing mitochondrial membrane potential decrease. For samples in 6-well plates, this kit can detect 100 samples; for samples in 12-well plates, this kit can detect 200 samples.

Product Features

1. Ratiometric Dual-Emission Probe: JC-1 forms red fluorescent J-aggregates in polarized mitochondria and green fluorescent monomers in depolarized mitochondria, enabling ratiometric analysis.

2. Early Apoptosis Detection: Mitochondrial membrane potential loss is an early event in apoptosis; JC-1 detects ¦¤¦·m changes before other apoptotic markers.

3. Positive Control Included: CCCP (10 mM) is provided as a positive control for inducing mitochondrial depolarization.

4. Multiple Detection Platforms: Suitable for flow cytometry, fluorescence microscopy, laser confocal microscopy, and fluorescence microplate reader.

5. Complete Kit Format: Includes JC-1 (200¡Á), ultrapure water, JC-1 staining buffer (5¡Á), and CCCP (10 mM).

Specifications

Size: 100 tests

Components:

JC-1 (200¡Á): 100 µL per tube, 5 tubes total

Ultrapure Water: 90 mL

JC-1 Staining Buffer (5¡Á): 80 mL

CCCP (10 mM): 50 µL

Detection Wavelength: JC-1 monomer Ex 514 nm/Em 529 nm; J-aggregate Ex 585 nm/Em 590 nm

Application: Detection of mitochondrial membrane potential changes in cells, tissues, or purified mitochondria

Storage and Stability

Storage Conditions: Store at -20¡ãC for 12 months from the date of manufacture. JC-1 (200¡Á) must be protected from light and avoid repeated freeze-thaw cycles. Ultrapure water and JC-1 Staining Buffer (5¡Á) may be stored at 2-8¡ãC.

Shelf Life: 12 months from the date of manufacture when stored as directed.

Protocol (For Reference Only)

Important: JC-1 is light-sensitive; protect from light during storage and handling. Prepare JC-1 working solution fresh before use. Analyze samples on ice and within 30 minutes after staining.

1. Preparation of JC-1 Working Solution:

For each well of a 6-well plate, 1 mL of JC-1 working solution is required; adjust volumes for other culture vessels accordingly. For cell suspensions, 0.5 mL of JC-1 working solution is required per 500,000-1,000,000 cells. Take an appropriate volume of JC-1 (200¡Á) and dilute with ultrapure water at a ratio of 50 µL JC-1 (200¡Á) to 8 mL ultrapure water. Vortex vigorously to dissolve and mix thoroughly, then let stand at room temperature for 1-2 minutes to ensure complete dissolution. Then add 2 mL of JC-1 Staining Buffer (5¡Á) and mix to obtain JC-1 working solution.

2. Positive Control Setup:

Dilute CCCP (10 mM) in cell culture medium at 1:1,000 to 1:100 depending on cell type and treat cells for 20 minutes. Then load JC-1 and detect mitochondrial membrane potential as described below. For most cells, treatment with 10 µM CCCP for 20 minutes completely abolishes mitochondrial membrane potential, and JC-1 staining appears green; normal cells stained with JC-1 appear red. CCCP may be added simultaneously with JC-1, but optimal concentration and treatment time may vary for specific cells and should be determined with reference to the literature.

3. For Suspension Cells:

1) Take 100,000-600,000 cells and resuspend in 0.5 mL cell culture medium; the medium may contain serum and phenol red.

2) Add 0.5 mL JC-1 working solution and mix by inverting several times. Incubate at 37¡ãC in a cell culture incubator for 20 minutes.

3) During incubation, prepare JC-1 Staining Buffer (1¡Á) by adding 4 mL ultrapure water per 1 mL JC-1 Staining Buffer (5¡Á), and place on ice.

4) After incubation at 37¡ãC, centrifuge at 600 x g for 3-4 minutes at 4¡ãC to pellet cells. Discard supernatant, taking care not to aspirate cells.

5) Wash twice with pre-chilled JC-1 Staining Buffer (1¡Á): add 1 mL JC-1 Staining Buffer (1¡Á) to resuspend cells, centrifuge at 600 x g for 3-4 minutes at 4¡ãC, discard supernatant. Repeat once.

6) After two washes, resuspend in an appropriate volume of JC-1 Staining Buffer (1¡Á) and analyze by flow cytometry, fluorescence microscopy, or laser confocal microscopy.

4. For Adherent Cells:

Note: For adherent cells, if flow cytometry analysis is desired, cells may be collected and resuspended first, then processed as described for suspension cells.

1) For one well of a 6-well plate, aspirate culture medium, wash once with PBS or other appropriate solution if necessary, and add 1 mL cell culture medium. The medium may contain serum and phenol red.

2) Add 1 mL JC-1 working solution and mix thoroughly. Incubate at 37¡ãC in a cell culture incubator for 20 minutes.

3) During incubation, prepare JC-1 Staining Buffer (1¡Á) by adding 4 mL ultrapure water per 1 mL JC-1 Staining Buffer (5¡Á), and place on ice.

4) After incubation at 37¡ãC, aspirate supernatant and wash twice with pre-chilled JC-1 Staining Buffer (1¡Á).

5) Add 2 mL cell culture medium, which may contain serum and phenol red.

6) Observe under a fluorescence microscope or laser confocal microscope.

5. For Purified Mitochondria:

1) Dilute the prepared JC-1 working solution 5-fold with JC-1 Staining Buffer (1¡Á).

2) Add 0.1 mL of purified mitochondria (10-100 µg total protein) to 0.9 mL of 5-fold diluted JC-1 working solution.

3) Detection by fluorescence spectrophotometer or fluorescence microplate reader: After mixing, perform time scan directly using a fluorescence spectrophotometer with excitation at 485 nm and emission at 590 nm. If using a fluorescence microplate reader and excitation cannot be set at 485 nm, set excitation within 475-520 nm. Alternatively, refer to the wavelength settings in step 6 below.

4) Observation by fluorescence microscopy or laser confocal microscopy: Follow the procedure in step 6 below.

6. Fluorescence Observation and Result Analysis:

For detection of JC-1 monomer, set excitation at 490 nm and emission at 530 nm; for detection of JC-1 aggregates, set excitation at 525 nm and emission at 590 nm. Note: It is not necessary to set excitation and emission at the maximum wavelengths for fluorescence measurement.

If using a fluorescence microscope, detection of JC-1 monomer may reference settings for observing other green fluorescence, such as GFP or FITC; detection of JC-1 aggregates may reference settings for observing other red fluorescence, such as propidium iodide or Cy3. Green fluorescence indicates decreased mitochondrial membrane potential and the cell is likely in early apoptosis. Red fluorescence indicates relatively normal mitochondrial membrane potential and normal cell state.

Precautions

1. JC-1 (200¡Á) may be warmed in a 20-25¡ãC water bath until completely dissolved before use. Briefly centrifuge before opening to collect liquid from the inner wall of the cap.

2. JC-1 is light-sensitive; protect from light during storage and handling.

3. When preparing JC-1 working solution, JC-1 (200¡Á) must first be fully dissolved and vortexed in the ultrapure water provided by the kit before adding JC-1 Staining Buffer (5¡Á). If JC-1 Staining Buffer (1¡Á) is prepared first and then JC-1 (200¡Á) is added, JC-1 will be difficult to dissolve completely, seriously affecting subsequent detection.

4. Samples should be kept on ice and analyzed as soon as possible, preferably within 30 minutes after staining. Do not dilute the entire JC-1 Staining Buffer (5¡Á) to JC-1 Staining Buffer (1¡Á); JC-1 Staining Buffer (5¡Á) must be used directly during the procedure.

5. If precipitate is observed in JC-1 Staining Buffer (5¡Á), it must be completely dissolved before use; heating at 37¡ãC may be used to promote dissolution.

6. CCCP is a mitochondrial electron transport chain inhibitor and is harmful to humans. Handle with care and take appropriate protective measures to avoid direct contact or inhalation.

7. This product is for scientific research by professionals only and is not intended for clinical diagnosis or treatment, food, or drugs. Do not store in ordinary residential areas.

8. For your safety and health, wear laboratory protective clothing and disposable gloves during operation.

Disclaimer

1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.

2. Due to the variable nature of biological research, optimization of staining conditions is recommended for specific cell types and experimental systems.

3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.

4. Wear appropriate protective clothing and gloves when handling this product. CCCP is a mitochondrial uncoupler and toxic; review the Safety Data Sheet before handling.

Ordering Information

Catalog Number: IC-6568

Product Name: Mitochondrial Membrane Potential Assay Kit (JC-1)

Size: 100 tests

Suggested Retail Price: CNY ¥1650.00 / USD $165.00 / EUR €198.00 / JPY ¥29700.00

Note: The specific price is subject to the transaction price in the offline contract. This document is provided for technical reference only and does not constitute any form of sales advertisement or invitation to offer. For product details and purchase inquiries, please contact local sales representatives.