Di-4-ANEPPDHQ 

Di-4-ANEPPDHQ is a polarity-sensitive membrane probe with a doubly positive charge that can be used to visualize membrane microdomains in living Arabidopsis cells. Di-4-ANEPPDHQ produces a fluorescence spectrum shift by changing the polarity of the lipid environment after embedding into the cell membrane, thereby quantifying the degree of lipid accumulation. The excitation wavelength of Di-4-ANEPPDHQ is 488 nm, and the emission wavelengths are 560 nm and 650 nm in the liquid ordered and disordered phases, respectively. Di-4-ANEPPDHQ is often used for fluorescence imaging studies of membrane microstructure, lipid order, and membrane tension response in living cells[1][2][3].

I. Arabidopsis thaliana live cell staining^[1]
Steps: 1. Sample preparation: 3-day-old Arabidopsis seedlings were placed in 1/2 MS medium, and root hairs and epidermal cells were taken for staining.
2. Staining operation: Add 5 μM Di-4-ANEPPDHQ (dissolved in 1/2 MS medium), incubate on ice for 5 minutes, and wash with cold 1/2 MS medium for 1 minute.
3. Plasmolysis verification: Treat with 650 mM Sorbitol (HY-B0400) for 5 minutes to confirm that the dye labels the cell membrane rather than the cell wall.
4. Fluorescence detection: using a confocal microscope (Leica SP5), 488 nm laser excitation, dual-channel acquisition: green channel 500-580 nm, red channel 620-750 nm. 5. GP value calculation: using ImageJ macro program to process images.

II. Detection of A549 cell membrane tension response^[2]
Steps:
1. Cell culture: A549 cells were cultured to confluence or non-confluence, digested with trypsin (HY-B2118) and plated on glass-bottom culture dishes.
2. Osmotic pressure treatment:
Hyperosmotic pressure: Add medium containing 100 mM sucrose and incubate for 10 minutes;
Hypoosmotic pressure: Mild hypotonic pressure (25% 1 mM CaCl₂+75% medium) or severe hypotonic pressure (50% 1 mM CaCl₂ + 50% medium) treatment for 10 minutes.
3. Staining operation: Add 1-5 μM Di-4-ANEPPDHQ and incubate at room temperature for 1-30 minutes (short-term labeling of cell membranes, long-term visualization of endosomes).
4. Fluorescence detection: Excitation wavelength 488 nm, emission channels 500-580 nm and 620-750 nm, use Zeiss 780 microscope to collect spectral images.
5. Data analysis: Analyze lipid accumulation by changes in GP value. The decrease in GP value after hypotonic treatment indicates that lipids are loosely arranged.

III. Liposomes and giant plasma membrane vesicles (GPMVs)^[3]
Steps:
1. Liposome preparation:
POPC and cholesterol are dissolved in chloroform in proportion, evaporated into film under nitrogen flow, and vacuum dried overnight;
HEPES buffer (10 mM HEPES (HY-D0857), 150 mM NaCl, pH 7.0) is added for hydration for 30 minutes, vortexed and extruded through a 100 nm polycarbonate membrane, with a lipid concentration of 0.5 mM.
2. Dye incorporation: Di-4-ANEPPDHQ (aqueous solution) was added to the hydrated liposomes at a molar ratio of 1:100, with a final concentration of about 10 μM.
3. GPMVs preparation: RBL-2H3 cells were treated with 25 mM paraformaldehyde (HY-Y0333) + 10 mM DTT (HY-15917) at 37°C for 1 hour, collected and stained with 250 nM Di-4-ANEPPDHQ for 30 minutes.
4. Spectral detection:
Steady-state spectroscopy: Fluorolog-3 spectrometer, 488 nm excitation, emission range 500-700 nm;
Time-resolved spectroscopy (TRES): 470 nm pulsed laser excitation, detection of 540-700 nm emission, analysis of fluorescence decay kinetics.

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

665.54

C₃₂H₄₇Br₂N₃O₂

797785-10-5

Solid

Brown to reddish brown

635

465

CCCCN(C1=CC(C=C2)=C(C=C1)C=C2/C=C/C(C=C3)=CC=[N+]3CC(O)C[N+](C)(C)CCO)CCCC.[Br-].[Br-]

Room temperature in continental US; may vary elsewhere.

* 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. When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

• [1]. Xiaoyu Zhao, et al. Di-4-ANEPPDHQ, a fluorescent probe for the visualisation of membrane microdomains in living Arabidopsis thaliana cells. Plant Physiol Biochem. 2015 Feb;87:53-60.  [Content Brief]

  [2]. Li N, et al. Di-4-ANEPPDHQ probes the response of lipid packing to the membrane tension change in living cells[J]. Chinese Chemical Letters, 2022, 33(3): 1377-1380.

  [3]. Amaro M, et al. Laurdan and Di-4-ANEPPDHQ probe different properties of the membrane. J Phys D Appl Phys. 2017 Apr 5;50(13):134004.  [Content Brief]