DiOC14(3) (hydroxyethanesulfonate)

Operating Instructions (The following is a recommended experimental protocol for guidance only, and adjustments should be made according to your specific requirements)

1. Stock Solution Preparation
1.1 Solvent: PBS can be used to prepare the stock solution.

2. Working Solution Preparation
2.1 Diluent: Complete medium.
2.2 Working Concentration:
2.2.1 Live Cell Membrane Staining: Dilute into growth medium at a ratio of 1:2000^[3].
2.2.2 Lipid Bilayer Membrane Staining: Approximately 10 μg/mL, dissolved in PBS^[1].
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample Type Description
3.1.1 Adherent Cells: Aspirate the medium from the culture wells and add growth medium containing the dye.
3.1.2 Lipid Bilayer Membranes: Immerse the lipid bilayer membranes on coverslips into PBS solution containing the dye.
3.2 Incubation Conditions
3.2.1 Live Cell Staining: Incubate in a 37 °C, 5% CO₂ incubator in the dark for 30-40 min.
3.2.2 Lipid Bilayer Membrane Staining: Incubate in PBS at room temperature.
3.3 Washing Step: After incubation, aspirate the dye-containing medium/PBS, and wash the samples twice with pre-warmed PBS to remove unbound free dye and reduce background fluorescence.

4. Control Setup
4.1 Blank Control: Incubate cell-free scaffolds and BMCP samples in complete medium for the same duration.

5. Detection and Analysis
5.1 Instrument Type: Fluorescence microscope or confocal microscope.
5.2 Excitation/Emission Wavelengths: Excitation wavelength of 477 nm and emission wavelength of 525 nm; detection can also be performed with excitation at 488 nm and emission at 500-550 nm
5.3 Result Analysis: After the dye inserts into the lipid bilayer of the cell membrane, the cell membrane shows bright green fluorescence, which can clearly display the cell outline, morphology and protrusions. Fluorescence is localized to the cell membrane, with no obvious fluorescence in the cytoplasm and nucleus.

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