DiSC₃(5) targets cell membranes and is sensitive to membrane potential changes. The compound does not target a specific protein or enzyme but rather partitions into membranes based on its lipophilic cationic properties. In hyperpolarized membranes, the dye accumulates, leading to fluorescence quenching. Upon membrane depolarization, the dye is released from cells, resulting in increased fluorescence intensity. This property allows DiSC₃(5) to be used as a probe for monitoring membrane potential changes.

1. Preparation of DiSC3 (5) membrane staining solution:
1.1 Preparation of DMSO or EtOH stock solution: The stock solution should be prepared in DMSO or EtOH to a concentration of 1-5 mM.
Note 1): The unused portion of the stock solution should be stored at -20 ℃. Avoid repeated freezing/thawing cycles.
1.2 Preparation of working solution: Dilute the stock solution into a suitable buffer solution, such as serum-free medium, HBSS, or PBS, to prepare a working solution of 1 to 5 uM.
Note 2): For different cell types and/or experimental conditions, the concentration of the working solution should be determined based on your specific needs.

2. Stain the cells into a suspension:
2.1 The cell density suspended in the dye working solution is 1 × 10~6/mL.
2.2 Incubate at 37 ° C for 2-20 minutes. The incubation time depends on the cell types. Firstly, incubate for 20 minutes, and then optimize as needed to obtain uniform labeling.
2.3 Centrifuge the marked suspension tube at 1000 to 1500rpm for 5 minutes.
2.4 Remove the supernatant and gently resuspend the cells in preheated (37 ° C) growth medium.
Wash twice according to steps 2.3 and 2.4.

3. Stained adherent cells:
3.1 Culture adherent cells on sterile glass coverslips.
3.2 Remove the cover glass from the growth medium and gently drain excess medium. Place the cover glass in the humidity chamber.
3.3 Transfer 100 μ L of dye working solution to the corner of a cover glass and gently stir until all cells are covered.
3.4 Incubate the cover glass at 37 ° C for 2-20 minutes. The cultivation time varies depending on the cell type. Start incubating for 20 minutes, then optimize as needed to obtain uniform labeling.
3.5 Discharge the dye working solution and clean the cover glass with growth medium two to three times. For each washing cycle, cover the cells with preheated growth medium, incubate for 5-10 minutes, and then drain the medium.

4. Microscopic examination.

5. Flow cytometry detection:
Cells labeled with DiS can be analyzed using a conventional FL3 flow cytometer.

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The in vitro activity of DiSC₃(5) is its function as a membrane potential-sensitive fluorescent probe. The compound is a lipophilic cationic dye that accumulates in hyperpolarized cell membranes, causing fluorescence quenching. When the membrane depolarizes, the dye is released from cells and fluorescence intensity increases. This enables real-time monitoring of mitochondrial membrane potential changes and membrane hyperpolarization. The dye has excitation/emission maxima of 622/670 nm.

DiSC₃(5) is not used as a therapeutic agent. Its in vivo utility is limited to research applications involving the measurement of membrane potential in cells and mitochondria. The compound is not intended for systemic therapeutic use.

3,3'-Dipropyl-2,2'-thiadicarbocyanine iodide [DiS-C(3)(5)], often used as a tracer dye to assess the mitochondrial membrane potential, was investigated in detail regarding its effects on the structure and function of isolated mitochondria. As reported previously, DiS-C(3)(5) had an inhibitory effect on NADH-driven mitochondrial electron transfer. On the contrary, in the presence of inorganic phosphate, DiS-C(3)(5) showed dose-dependent biphasic effects on mitochondria energized by succinate. At higher concentrations, such as 50 micro m, DiS-C(3)(5) accelerated mitochondrial oxygen consumption. Measurements of the permeability of DiS-C(3)(5)-treated mitochondrial membranes to poly(ethylene glycol) and analysis of mitochondrial configuration by transmission electron microscopy revealed that the accelerating effect of DiS-C(3)(5) on mitochondrial oxygen consumption reflects the induction of the mitochondrial permeability transition (PT). When the mitochondrial PT was induced by DiS-C(3)(5), release of mitochondrial cytochrome c was observed, as in the case of the PT induced by Ca(2+). On the contrary, at a low concentration such as 5 micro m, DiS-C(3)(5) showed an inhibitory effect on the latent oxygen consumption by mitochondria. This effect was shown to reflect inhibition of the PT induced by a low concentration of Ca(2+). Furthermore, in the absence of inorganic phosphate, DiS-C(3)(5) caused mitochondrial swelling. Under this condition, DiS-C(3)(5) caused changes in the membrane status of the mitochondria, but did not induce a release of mitochondrial cytochrome c [1].

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In vitro membrane potential assays for DiSC₃(5) involve incubating cells or isolated mitochondria with the fluorescent probe. The dye accumulates in membranes based on the membrane potential. Fluorescence is measured using a fluorescence spectrophotometer or microplate reader with excitation at 622 nm and emission at 670 nm. Changes in fluorescence intensity reflect changes in membrane potential. The assay can be used to study the effects of drugs or other treatments on mitochondrial function.

For in vitro cellular experiments, DiSC₃(5) is dissolved in DMSO and diluted in cell culture medium. Cells are incubated with the dye, and fluorescence is monitored in real time using a fluorescence microscope or plate reader. The dye is used to assess mitochondrial membrane potential and monitor membrane hyperpolarization.

In vivo animal experiments with DiSC₃(5) are not typically performed, as the compound is primarily used as an in vitro probe for membrane potential measurements.

Pharmacokinetic properties of DiSC₃(5) have not been characterized, as the compound is a research-use fluorescent probe rather than a drug candidate. The compound has a molecular weight of 546.53 g/mol and should be stored under appropriate conditions, protected from light.

DiSC₃(5) is intended for research use only and not for human therapeutic or diagnostic applications. Standard laboratory safety precautions should be observed when handling this chemical reagent.

[1]. Multiple effects of DiS-C3(5) on mitochondrial structure and function. Eur J Biochem. 2004;271(17):3573-3579.

[2]. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles. Biochemistry. 1974 Jul 30;13(16):3315-30.

[3]. The effect of the fluorescent probe, 3,3'-dipropylthiodicarbocyanine iodide, on the membrane potential of Ehrlich ascites tumor cells. Biochem Biophys Res Commun. 1980 Jul 31;95(2):722-7.

See also: 3,3'-dipropyl-2,2'-thiodicarbocyanine (preferred).

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DiSC₃(5) (3,3'-Dipropylthiadicarbocyanine iodide) is a lipophilic cationic fluorescent probe sensitive to membrane potential changes. It is used to assess mitochondrial membrane potential and monitor membrane hyperpolarization in real time. The compound has no clinical or therapeutic applications.

C25H27IN2S2

546.53

546.066

C, 54.94; H, 4.98; I, 23.22; N, 5.13; S, 11.73

53213-94-8

5706741

Blue to dark blue solid powder

245ºC (dec.)(lit.)

3.36

0

4

7

30

564

0

CCCN\1C2=CC=CC=C2S/C1=C/C=C/C=C/C3=[N+](C4=CC=CC=C4S3)CCC.[I-]

GDEURKKLNUGTDA-UHFFFAOYSA-M

InChI=1S/C25H27N2S2.HI/c1-3-18-26-20-12-8-10-14-22(20)28-24(26)16-6-5-7-17-25-27(19-4-2)21-13-9-11-15-23(21)29-25;/h5-17H,3-4,18-19H2,1-2H3;1H/q+1;/p-1

(2E)-3-propyl-2-[(2E,4E)-5-(3-propyl-1,3-benzothiazol-3-ium-2-yl)penta-2,4-dienylidene]-1,3-benzothiazole;iodide

3,3'-Dipropylthiadicarbocyanine iodide; 53213-94-8; Disc3(5); Dptdci; Dis C3-(5); 3,3'-Dipropylthiodicarbocyanine iodide; 3,3'-dipropyl-2,2'-thiadicarbocyanine; Di-S-C3-5; NK 2251;

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: 16.67 mg/mL (30.50 mM)

Solubility in Formulation 1: 1.25 mg/mL (2.29 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 (2.29 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.)