Dehydroergosterol

Guidelines (The following is a recommended operating procedure for reference only, which needs to be adjusted according to your specific requirements)

1. Preparation of Working Solution
1.1 Diluent: Cell culture medium; Alternative diluents: PBS, bicarbonate-buffered Ham's F-12 medium supplemented with 5% lipoprotein-deficient serum (LPDS).
1.2 Working concentration: 12.5 μM; Alternative working concentrations/formulations: 20 μg Dehydroergosterol per 1 mL cell culture medium, 20 μg Dehydroergosterol per sample (delivered as DHE-MbCD complex), POPC:DHE at a molar ratio of 65:35 (present in large unilamellar vesicles).
1.3 Notes: Adjust the concentration of the working solution as needed; Prepare it immediately before use.

2. Staining Procedure
2.1 Description of Sample Types
2.1.1 Adherent cultured cells^[1][2]:
2.1.1.1 L cell fibroblasts[1]: Cells are cultured on chamber coverslips; No trypsin digestion step is mentioned for staining.
2.1.1.2 CHO cell line TRVb-1/Tac-TGN38[2]: Trypsin digestion is required after incubation.
2.2 Incubation Conditions
2.2.1 Adherent cultured cells^[1][2]:
2.2.1.1 Stock solution diluted with medium (ethanol-based system)[1]: Incubate for 2 days, with temperature unspecified.
2.2.1.2 Large unilamellar vesicle system[1]: Incubate for 1 day, with temperature unspecified.
2.2.1.3 DHE-MbCD complex system[1]: Incubate at room temperature for 45 min; Protect the complex from light during preparation.
2.2.1.4 Bicarbonate-buffered Ham's F-12 medium system supplemented with 5% LPDS[2]: Incubate cells in a humidified incubator at 37°C with 5% CO₂ for 16-20 h.
2.3 Washing Steps
2.3.1 Adherent cultured cells^[1][2]:
2.3.1.1 Stock solution diluted with medium (ethanol-based system)[1]: Wash cells multiple times with an unspecified buffer.
2.3.1.2 DHE-MbCD complex system[1]: Wash cells 3 times with PBS.
2.3.1.3 CHO cell line TRVb-1/Tac-TGN38[2]: Wash the cell monolayer twice with PBS after incubation; After trypsin digestion, resuspend the cells by centrifugation and wash twice with PBS again.
2.4 Post-streatment
2.4.1 CHO cell line TRVb-1/Tac-TGN38[2]: Trypsinize the labeled cells at 37°C for 5 min, centrifuge at approximately 135 × g, resuspend in Ham's F-12 medium containing 5% LPDS, and seed onto coverslips coated with poly-D-lysine or fibronectin; Continue culturing the cells for 16-20 h before imaging.

3. Control Setup
3.1 Set up unlabeled cell controls to evaluate autofluorescence under the same imaging conditions.

4. Detection and Analysis
4.1 Instrument types: Multiphoton laser scanning microscope; Fluorescence microscope equipped with a cooled CCD camera.
4.2 Excitation/Emission Wavelengths
4.2.1 Multiphoton laser scanning microscope[1]: Excitation wavelength: 900 nm; Detect emitted light using D400/100 and UV 440LP dichroic filters.
4.2.2 Monomeric DHE^[1][2]: Maximum excitation wavelengths: 311 nm, 324 nm, 340 nm; Maximum emission wavelengths: 354 nm, 371 nm, 375 nm, 390 nm, 394 nm, 414 nm.
4.2.3 Crystalline DHE[1]: Maximum emission wavelengths: 404 nm, 426 nm.
4.2.4 Fluorescence microscope with UV filter set[2]: Excitation filter: 335 nm (20 nm bandpass); Dichroic filter: 365 nm longpass; Emission filter: 405 nm (40 nm bandpass).
4.3 Result Analysis
4.3.1 Fluorescence Intensity and Morphology^[1][2]:
4.3.1.1 Crystalline DHE shows strong, bright patchy fluorescence; Monomeric DHE shows moderate-intensity uniform fluorescence.
4.3.1.2 After photobleaching, the DHE fluorescence in the bleached area recovers within 20 min at 37°C, indicating dynamic sterol trafficking.
4.3.2 Fluorescence Localization^[1][2]:
4.3.2.1 DHE from ethanol stock solution[1]: Initially accumulates in lysosomes as crystalline DHE; After 2-3 days, monomeric DHE distributes to the plasma membrane, lipid droplets, endoplasmic reticulum and other organelle membranes.
4.3.2.2 DHE from large unilamellar vesicles[1]: Monomeric DHE distributes to the plasma membrane and intracellular compartments, with partial accumulation in lipid storage vesicles.
4.3.2.3 DHE from DHE-MbCD complex[1]: Monomeric DHE strongly labels the plasma membrane (including microvilli and filopodia), with weak labeling of other intracellular membranes.
4.3.2.4 DHE in CHO cell line TRVb-1/Tac-TGN38[2]: Localizes to the plasma membrane and perinuclear regions, including the endosomal recycling compartment (ERC) and trans-Golgi network (TGN).
4.3.3 Fluorescence Distribution[1]: In the plasma membrane of living cells, DHE distributes non-randomly in sterol-rich and sterol-poor regions.
4.3.4 Ratio Analysis[1]: Calculate the ratio of fluorescence intensity at 426 nm to that at 373 nm to distinguish crystalline DHE (ratio approximately 3.4) from monomeric DHE (ratio approximately 0.3).
4.3.5 Colocalization Analysis[2]: DHE shows extensive colocalization with endocytosed transferrin (ERC marker) and Tac-TGN38 (TGN marker).

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