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) in isolated rabbit hearts, changes cardiac electrical activity, such as significantly lows down heart rate and ventricular conduction, and decreases ischemia detection ability^[1]. In experiments on cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM), Di-4-ANEPPS (6 μM; 1 min), can be used to evaluate the electrophysiological characteristics of hiPSC-CM and detect the effect of drugs on its electrophysiology^[2].

Di-4-ANEPPS is applicable for various experimental setups as follows^[1][2][3]:
1. Rabbit Isolated Heart Staining Method^[1]:
Experimental subjects are randomly divided into staining and control groups.
After deeply anesthetizing the rabbits, tracheal intubation and artificial ventilation are performed, followed by thoracotomy to remove the heart which is then placed in cold Krebs-Henseleit (K-H) solution.
The aorta is cannulated, and the heart is placed in a bath filled with K-H solution. The heart is perfused with K-H solution saturated with mixed gas (95% O₂ and 5% CO₂) under stable conditions (pressure 80 mmHg, 37°C) for stabilization for 20 minutes.
In the staining group, after the stabilization period, the heart is perfused with a 2 μM Di-4-ANEPPS solution for 20 minutes, followed by rinsing with K-H solution for 20 minutes to wash away unbound dye molecules.
The control group does not undergo dye loading and rinsing steps, only extending the stabilization period to 60 minutes. Throughout the experiment, electrograms (EG) are recorded non-invasively through bipolar leads from Ag-AgCl disc electrodes placed in the bath, while coronary artery flow is measured periodically to monitor overall stability of the heart preparation. EG is sampled at a rate of 2 kHz with 16-bit resolution throughout the experiment.

2. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CM) Staining Experiment^[2]:
hiPSC-CMs are stored in liquid nitrogen and thawed before use.
Cells are cultured on 96-well glass-bottom plates pre-coated with fibronectin, incubated in a humidified incubator at 37°C for 3 hours.
Before the experiment, cells are washed with serum-free medium (SF media).
Then, hiPSC-CMs are stained with 6 μM Di-4-ANEPPS for 1 minute at room temperature, followed by washing with SF media without indicator and incubating for 2 hours before experiments.
Plates are placed in an environmental-controlled incubator (37°C, 5% CO₂), recording fluorescence signals of Di-4-ANEPPS in a 0.2mm×0.2mm area under a 40X objective (excitation wavelength 470±10 nm).
Note: Light-emitting diodes (LEDs) can be used as light sources, 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]:
Brain slices are obtained from transgenic mice aged 25-90 days.
During the experiment, mice are deeply anesthetized with isoflurane, decapitated, and the head immersed in 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).
A 300 μm coronal slice is taken from the frontal parietal cortex, first incubated at 37°C for 30 minutes, then kept at room temperature.
Acute brain slices are transferred onto an upright microscope (10× objective, 0.3 NA) and perfused with physiological saline bubbled with mixed gas (5% CO₂/95% O₂). All experimental measurements are conducted at 34°C.
Synaptic stimulation is performed using a stimulus isolation unit; stimulating electrodes (1.5 mm borosilicate glass with fine filament, resistance ~2MΩ) are backfilled with physiological saline. Initially, three synaptic stimulations are applied at intervals of 120 ms (8.3 Hz), followed by another three synaptic stimulations at intervals of 12 ms (83 Hz) 1 second later.
Each optical experiment includes two sets of synaptic triplets lasting 3 seconds (3 seconds of light exposure), with at least 12 seconds of darkness (no light exposure) between consecutive scans. The light source for Di-4-ANEPPS is LED GYR 500-600 nm, with an optical filter set including excitation light 520/60 nm, dichroic mirror 570 nm, and emission light 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)

• [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]