NRGolgi

Operating Instructions
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 Most dyes are typically dissolved in organic solvents, such as anhydrous DMSO.
1.2 Concentration recommendation: Prepare a high-concentration stock solution at 1-10 mM.

2. Working Solution Preparation
2.1 Diluent: HBSS.
2.2 Working concentration: 2.5 μM (as a 1:1 molar ratio conjugate with BSA) for colocalization experiments; 5 μM (as a BSA conjugate) for ratiometric imaging experiments.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample type: Adherent cells (KB and HeLa cells)^[1]
3.1.1 Adherent cells: No trypsin digestion required; use cells seeded on chamber coverslips.
3.2 Incubation conditions: Use NRGolgi_E working solution at a concentration of 2.5 μM (BSA conjugate) or 5 μM (BSA conjugate); incubate the cells at 4 °C for 30 min, followed by washing with pre-warmed HBSS, and then continue incubation at 37 °C for 30 min.
3.3 Washing steps: Wash the cells twice with pre-cooled HBSS before incubation; wash with pre-warmed HBSS after incubation at 4 °C.

4. Control Setup
4.1 Set up colocalization controls using BODIPY FL C5-ceramide (a commercial Golgi marker) at a concentration of 2.5 μM (as a 1:1 molar ratio conjugate with BSA) or 150 nM (dissolved in 1 mM β-cyclodextrin solution).

5. Detection and Analysis
5.1 Instrument: Confocal microscope.
5.1.1 Excitation wavelength: 488 nm; detect emission wavelengths in the ranges of 550-600 nm and 600-650 nm.
5.2 Result analysis:
5.2.1 Fluorescence intensity change: The green/red fluorescence intensity ratio (I_550-600/I_600-650) decreases with increased local polarity (decreased lipid order) and increases with decreased local polarity (increased lipid order).
5.2.2 Fluorescence localization: Selectively localizes to the Golgi apparatus in live cells.
5.2.3 Color change: Ratiometric pseudocolor shift reflects changes in local polarity; lower polarity (higher lipid order) corresponds to a higher ratio value (cyan pseudocolor), while higher polarity (lower lipid order) corresponds to a lower ratio value (orange-red pseudocolor).

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