JC-1 (CBIC2) is an ideal fluorescent probe extensively used to detect mitochondrial membrane potential. JC-1 dye accumulates in mitochondria in a potential-dependent manner and may be utilized to detect membrane potential in cells, tissues, or purified mitochondria. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form polymers, which emit strong red fluorescence (Ex=585 nm, Em=590 nm); when the mitochondrial membrane potential is low, JC-1 cannot aggregate in mitochondria. In the matrix, green fluorescence (Ex=514 nm, Em=529 nm) is produced.

Physicochemical Properties

Molecular Formula	C25H27CL4IN4
Molecular Weight	652.2235
Exact Mass	650.003
CAS #	3520-43-2
Related CAS #	3520-43-2;
PubChem CID	5492929
Appearance	Purple to purplish red solid powder
LogP	4.157
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	6
Heavy Atom Count	34
Complexity	625
Defined Atom Stereocenter Count	0
SMILES	CCN1C2=CC(=C(C=C2[N+](=C1/C=C/C=C3N(C4=CC(=C(C=C4N3CC)Cl)Cl)CC)CC)Cl)Cl.[I-]
InChi Key	FYNNIUVBDKICAX-UHFFFAOYSA-M
InChi Code	InChI=1S/C25H27Cl4N4.HI/c1-5-30-20-12-16(26)17(27)13-21(20)31(6-2)24(30)10-9-11-25-32(7-3)22-14-18(28)19(29)15-23(22)33(25)8-4/h9-15H,5-8H2,1-4H31H/q+1/p-1
Chemical Name	1H-Benzimidazolium, 5,6-dichloro-2-(3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl)-1,3-diethyl-, iodide
Synonyms	JC 1 JC-1 JC1
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	JC-1 staining solution composition 1.1 Make the stock solution with DMSO to achieve a concentration of 5 mg/mL of JC-1. For instance, dissolve 5 mg of JC-1 in 1 mL of DMSO. It is advised to aliquot the JC-1 storage solution and store it in the dark at -20°C or -80°C. 1.2 Working solution replacement: Make a JC-1 working solution at a concentration of 1–20 μg/mL by mixing PBS or previously prepared serum-free cell bone marrow with the storage solution. Note: 1) Please utilize the existing configuration and modify the JC-1's working liquid concentration in accordance with the real circumstances. 2) You can add 20% diluent Pluronic F127 solution to the working solution, with a final concentration of 0.02-0.05%, if the effect of JC-1 entering the cells is not good. Pluronic F127 can assist JC-1 in entering the cell by preventing it from aggregating in the buffer. JC-1 staining 1) Plate cells at a density of 5×105 cells/mL using a 6-well plate as an example. In an incubator with 5% CO2 and 37°C, cultivate overnight. Note: During induction, it is advised that the cell density not surpass 1×106/mL. Additionally, you can change the density to a density that suits your own culture type. 2) Once sterile, take 0.5 mL of the cell suspension. 3) Centrifuge for three to five minutes at 400 g; remove supernatant. 4) After resuspending the cells in 1 milliliter of JC-1 working solution, incubate for 15 to 30 minutes on the side wall of an incubator set at 37°C with 5% CO2. 5) After that, 6) resuspend the cells in 2 milliliters of buffer or cell culture media, and centrifuge for an additional 5 minutes at 400 g. Discard the supernatant and repeat twice. 7) Re-suspend the cells in 1 mL of new culture medium or buffer, and then go straight to the observation of fluorescence microscopy or flow cytometry analysis. 8) Data analysis (flow cytometry): Red JC-1 aggregates from healthy cell mitochondria are detected using the FL2 channel; green ones containing green Note: If the cells are to be used for microplate reader detection, resuspend them with 300 μL buffer and proceed as directed. Transfer the stained cells into a 100 μL volume of a light-tight 96-well plate, and then carry out fluorescence microplate analysis.
References	[1]. JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death Dis. 2012 Nov 22;3:e430. [2]. Ratiometric high-resolution imaging of JC-1 fluorescence reveals the subcellular heterogeneity of astrocytic mitochondria. Pflügers Archiv - European Journal of Physiology. 2011,462(5): 693-708. [3]. Real-time analysis of amyloid fibril formation of α-synuclein using a fibrillation-state-specific fluorescent probe of JC-1. Biochem. J. 2009, 418:311-323. [4]. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess delta psi changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 1997 Jul 7;411(1):77-82.
Additional Infomation	1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloroimidacarbocyanine iodide is a cyanine dye and an organic iodide salt. It has a role as a fluorochrome. It contains a 1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloroimidacarbocyanine.

Solubility Data

Solubility (In Vitro)

DMSO : ~5 mg/mL (~7.67 mM) H2O : < 0.1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: 1.25 mg/mL (1.92 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 1.25 mg/mL (1.92 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.5332 mL	7.6661 mL	15.3322 mL
	5 mM	0.3066 mL	1.5332 mL	3.0664 mL
	10 mM	0.1533 mL	0.7666 mL	1.5332 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.