Mitochondria
Mitochondria are the energy factories of the cell, the main site of intracellular oxidative phosphorylation and synthesis of adenosine triphosphate (ATP), which provides energy for cellular activities. 95% of the energy required for cellular life activities comes from mitochondria. In addition to supplying energy for cells, mitochondria are also involved in processes such as cell differentiation, cellular information transfer and apoptosis, and possess the ability to regulate cell growth and cell cycle. Therefore, assays such as mitochondria and mitochondrial membranes are an essential part of the study of cells.
Definition and importance of mitochondrial membrane potential
Mitochondrial membrane potential is the result of charge distribution on both sides of the inner mitochondrial membrane and is a key factor in maintaining normal mitochondrial function. In mitochondria, the electron transport chain generates a proton gradient through redox reactions that drive ATP synthesis. The stability of the mitochondrial membrane potential is directly related to the energy supply of the cell and its survival status. A large number of studies have shown that mitochondria are closely related to apoptosis, in which the decrease in mitochondrial transmembrane potential is considered to be one of the earliest events in the apoptotic cascade reaction process. It occurs before the appearance of apoptotic features (chromatin condensation, DNA breaks) in the nucleus, and once the mitochondrial transmembrane potential collapses, apoptosis is irreversible. Thus, the decrease in mitochondrial membrane potential is a hallmark event in the early stages of apoptosis.
Working Principle of JC-1 Probe
JC-1 is a cationic lipid fluorescent dye, which can be used as an indicator of mitochondrial transmembrane potential. JC-1 exists in two states, monomer and multimer. At low concentration, it exists as a monomer, and green fluorescence can be detected, and when detected by flow-through assay, it is usually in the FL-1 channel (the same channel as FITC). At high concentration, it exists as a multimer, and red light can be detected, and when detected by flow-through assay, it is usually in the FL-2 channel (the same channel as PE). JC-1 can also be used as a cationic lipid fluorescent dye, and can be used as a mitochondrial transmembrane potential indicator. and PE same channel). Due to the change of JC-1 concentration, a reversible transition process between monomer and multimer is formed.
In normal cells, when the membrane potential is normal, JC-1 enters the mitochondria through the mitochondrial membrane polarity and forms red fluorescent multimers due to the increase in concentration, while in apoptotic cells, the mitochondrial transmembrane potential is depolarized and JC-1 is released from the mitochondria at a reduced concentration and reversed to the green fluorescent monomer form. Therefore, changes in mitochondrial membrane potential can be detected by detecting green and red fluorescence.
Note: Images quoted from the internet
JC-10 Probe
JC-10, a derivative of JC-1, is a potential-dependent probe used to determine mitochondrial transmembrane potential by flow cytometry, fluorescence microscopy and microtiter plate-based fluorescence assays. In healthy cells, JC-10 selectively accumulates in mitochondria, generating red aggregates that exhibit a broad excitation spectrum and emission large values at 590 nm. However, in apoptotic and necrotic cells with low mitochondrial transmembrane potential, JC-10 diffuses out of the mitochondria and produces JC-10 monomers, resulting in a shift toward green emission (525 nm). It has been successfully used as an indicator of mitochondrial transmembrane potential in a variety of sample types including myocytes, neurons, intact tissues and isolated mitochondria.
JC-10--Replacement for JC-1
- Stability: JC-10 has less detection bias because it has higher solubility and higher sensitivity in aqueous media.
- Enhanced signal: JC-10 has a higher signal-to-background ratio than JC-1.
- Enhanced sensitivity:JC-10 is able to detect subtle changes in mitochondrial transmembrane potential loss better than JC-1 in all cell lines tested.
- Wide application:JC-10 can be used in primary rat hepatocytes.
- Convenient:JC-10 is compatible with fluorescent enzyme markers, cell imagers and flow cytometers.
Notes:. Mitochondrial membrane potential changes induced by comedones were measured in Jurkat cells with JC-10 and JC-1. After treating Jurkat cells with camptothecin (10 mM) for 4 h, JC-1 and JC-10 dye upsampling solutions were added to the wells and incubated for 30 min. The fluorescence intensity of the aggregated and monomeric forms of JC-1 and JC-10 was measured at Ex/Em = 490/525 nm and 490/590 nm using a NOVOstar enzyme labeler (BMG Labtech).
Analysis of results
When detecting JC-1 monomer, the excitation light can be set to 490 nm and the emission light can be set to 530 nm; when detecting JC-1 polymer, the excitation light can be set to 525 nm and the emission light can be set to 590 nm. Apoptosis was detected by flow cytometry, green fluorescence was detected through the FL-1 channel, and red fluorescence was detected through the FL-2 channel. FL-1+, FL-2 + are normal cells and FL-1+, FL-2- are apoptotic cells.
Note: The image source is the China Streamline Association website forums
Operate without doubt
Q: JC-1 Mitochondrial Membrane Potential Fluorescent Probe, can this product be used to stain tissue sections?
A: Digest the living tissue into individual cells to ensure the activity of the cells before staining.
Q: Can I fix the cells before staining with JC-1?
A: No. JC-1 dye accumulates in mitochondria in a potential-dependent manner and can be used to detect cells, tissues, or purified mitochondrial membrane potentials. Immobilization disrupts the electrical potential, resulting in the inability of the probe to accumulate on the mitochondria and loss of specificity.
Q:Â There are aggregated particles in the JC-1 working solution and I plan to remove them by centrifugation before staining, are there any recommended parameters for centrifugation?
A: The JC-1 Working Solution can be centrifuged at 13,000 g for about 1-2 minutes.
Ordering Information
|
Product Name |
Item No. |
Specification |
|
JC-1 mitochondrial membrane potential fluorescent probe |
40705ES03/08 |
1 mg/5 mg |
|
JC-10 mitochondrial membrane potential fluorescent probe |
40707ES03/08 |
1 mg/5 mg |
|
JC-1 Mitochondrial Membrane Potential Assay Kit |
40706ES60 |
100 tests |
|
JC-10 Mitochondrial Membrane Potential Assay Kit |
40752ES60 |
100 T |