ER-Fluidity

Operating Instructions (The following is a recommended experimental protocol for guidance only, and adjustments are required based on your specific needs).
1. ER-Fluidity (Compound 1) Stock Solution Preparation
1.1 Solvent: DMSO.
2. Working Solution Preparation
2.1 Diluent: 1× PBS containing β-Cyclodextrin (β-CD) (HY-107201).
2.2 Working concentration: 1.0 μM dye + 4.0 mM β-CD.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh prior to use.
3. Staining Procedure
3.1 Sample type: Adherent cells (HeLa cells)
3.1.1 Adherent cells: No trypsin digestion required; seed cells into glass-bottom dishes 1 day before imaging.
3.2 Incubation conditions: Incubate cells with the dye-β-CD complex (1.0 μM dye, 4.0 mM β-CD) at 37°C for 1 h.
3.3 Washing step: Omitted.
4. Detection and Analysis
4.1 Instrument: Confocal laser scanning microscope or multiphoton microscope.
4.1.1 Excitation/emission wavelengths: BODIPY group: Ex 488 nm, Em 510-600 nm; Coumarin (HY-N0709) group: Ex 740 nm (two-photon), Em 400-490 nm.
4.2 Result analysis:
4.2.1 Fluorescence intensity change: Under endoplasmic reticulum (ER) stress conditions, the decrease in ER membrane fluidity causes the fluorescence intensity of BODIPY to increase relative to that of Coumarin (HY-N0709).
4.2.2 Fluorescence localization: Fluorescence localizes to the endoplasmic reticulum (ER) membrane.
4.2.3 Fluorescence color: BODIPY emits green fluorescence; Coumarin emits yellow fluorescence.

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