Di-4-ANEPPS 

Di-4-ANEPPS is a voltage-sensitive dye that acts on voltage-gated ion channels (such as sodium channels) and inhibits sodium current, significantly reducing sodium current density, although specific values like IC₅₀ remain unclear. It mainly binds to the voltage-sensitive regions on the cell membrane, changing its fluorescence properties to reflect membrane potential changes and thus affecting the function of ion channels to exert its activity. This substance can be used in cardiovascular research, such as the electrophysiology of cardiomyocytes, myocardial ischemia, and the effects of drugs on cardiomyocytes. It is of great value in evaluating drug cardiotoxicity and exploring the mechanisms of arrhythmias^[1][2].

Di-4-ANEPPS (2 μM; 20 min) treatment of isolated rabbit hearts alters cardiac electrical activity, such as slowing heart rate and ventricular conduction, and reduces ischemia detection capability^[1].
Di-4-ANEPPS (6 μM; 1 min) can be used in experiments with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to evaluate the electrophysiological properties of hiPSC-CMs and to assess the effects of drugs on their electrophysiology^[2].
Guidelines (The following is our recommended experimental protocol. This protocol is for reference only, and specific operations should be adjusted according to your actual needs).
^[1][2][3]:
1. Isolated rabbit heart staining experiment^[1]:
Randomly divide the experimental subjects into a staining group and a control group.
After deep anesthesia of the rabbits, perform tracheal intubation and artificial ventilation, followed by thoracotomy to excise the heart and place it in cold Krebs-Henseleit (K-H) solution.
After aortic cannulation, place the heart into a bath filled with K-H solution and perfuse under stable conditions (pressure 80 mmHg, temperature 37°C) using K-H solution saturated with mixed gas (95% O₂ and 5% CO₂). Allow the heart to stabilize for 20 minutes.
After stabilization, perfuse the staining group hearts with 2 μM Di-4-ANEPPS solution for 20 minutes for staining, followed by washing with K-H solution for 20 minutes to remove unbound dye molecules.
The control group does not undergo dye loading or washing steps, but instead the stabilization period is extended to 60 minutes. Throughout the experiment, electrograms (EGs) are recorded non-contactly using bipolar leads from Ag-AgCl disc electrodes placed in the bath, while coronary flow is periodically measured to monitor the overall stability of the heart preparation.
Throughout the experiment, EGs are sampled at 2 kHz with 16-bit resolution.
2. Human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) staining experiment^[2]:
Store hiPSC-CMs in liquid nitrogen and revive them before use.
Cells are cultured on 96-well glass-bottom culture plates pre-coated with fibronectin and incubated in a humidified incubator at 37°C for 3 hours.
Before the experiment, wash the cells with serum-free medium (SF media).
Then stain hiPSC-CMs with 6 μM Di-4-ANEPPS at room temperature for 1 minute, followed by washing with SF media without indicator dye, and incubate in the incubator for 2 hours before experimentation.
Place the culture plate in an environmental control incubator (37°C, 5% CO₂) and record the fluorescence signal of Di-4-ANEPPS from a 0.2 mm × 0.2 mm region under a 40× objective lens (excitation wavelength 470±10 nm).
Note: A light-emitting diode (LED) may be used as the light source, and emitted light is collected by two photomultiplier tubes (PMTs) at 510-560 nm and 590-650 nm, respectively.
3. Mouse brain slice staining experiment^[3]:
Use brain slices obtained from transgenic mice aged 25-90 days.
During the experiment, deeply anesthetize the mice with isoflurane, then decapitate and remove the brain, immersing the head in ice-cold physiological saline (artificial cerebrospinal fluid, ACSF, containing 125 mM NaCl, 26 mM NaHCO₃, 2.3 mM KCl, 1.26 mM KH₂PO₄, 2 mM CaCl₂, 1 mM MgSO₄, and 10 mM glucose).
Cut 300 μm coronal slices from the frontal-parietal cortex, incubate first at 37°C for 30 minutes, and then keep at room temperature.
Transfer acute brain slices to an upright microscope (10× objective, 0.3 NA) and perfuse with physiological saline saturated with mixed gas (5% CO₂/95% O₂). All experimental measurements are conducted at 34°C.
Use a stimulus isolation unit for synaptic stimulation. The stimulation electrode (1.5 mm borosilicate glass with filament, resistance ~2 MΩ) is backfilled with physiological saline.
First apply three synaptic stimulations at a stimulus interval of 120 ms (8.3 Hz), then after 1 s apply another three synaptic stimulations at an interval of 12 ms (83 Hz).
Each optical experiment consists of two synaptic stimulation triplets lasting 3 seconds (3 seconds of light exposure), with at least a 12 s dark period (no light exposure) between consecutive experimental scans.
The light source for Di-4-ANEPPS is LED GYR 500-600 nm, and the optical filter set includes excitation 520/60 nm, dichroic mirror 570 nm, and emission 600 LP.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

480.66

C₂₈H₃₆N₂O₃S

90134-00-2

Solid

Brown to reddish brown

705

496

O=S(CCC[N+]1=CC=C(/C=C/C2=CC=C3C=C(N(CCCC)CCCC)C=CC3=C2)C=C1)([O-])=O

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Marina Ronzhina, et al. Di-4-ANEPPS Modulates Electrical Activity and Progress of Myocardial Ischemia in Rabbit Isolated Heart. Front Physiol. 2021 Jun 10;12:667065.  [Content Brief]

  [2]. Hortigon-Vinagre MP, et al. The Use of Ratiometric Fluorescence Measurements of the Voltage Sensitive Dye Di-4-ANEPPS to Examine Action Potential Characteristics and Drug Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Toxicol Sci. 2016 Dec;154(2):320-331.  [Content Brief]