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.

[1]. A Perelman, et al. JC-1: alternative excitation wavelengths facilitate mitochondrial membrane potential cytometry. Cell Death Dis. 2012 Nov 22;3:e430.
[2]. Vera C. Keil, et al. 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]. Jung-Ho LEE, et al. 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]. Salvioli S, et al. 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.

C25H27CL4IN4

652.2235

650.0035

3520-43-2

3520-43-2;

Typically exists as solids (or liquids in special cases) at room temperature

CC[N+]1=C(/C=C/C=C2N(CC)C3=CC(Cl)=C(Cl)C=C3N\2CC)N(CC)C4=CC(Cl)=C(Cl)C=C14.[I-]

FYNNIUVBDKICAX-UHFFFAOYSA-M

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

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

JC 1 JC-1 JC1

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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

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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)