Di-4-ANEPPDHQ (solution)

Guide (The following is our recommended protocol. This protocol is only a guide and should be modified according to your specific needs).
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.