NRLD

Operating Instructions
(The following is a recommended experimental protocol for guidance only; adjust according to actual requirements)

1. Stock Solution Preparation
1.1 Recommended concentration: 1 mM.

2. Working Solution Preparation
2.1 Diluent: Pre-warmed HBSS.
2.2 Working concentration: 50 nM.
2.3 Notes: Adjust the working solution concentration as needed; prepare and use immediately.

3. Staining Procedure
3.1 Sample type: Adherent cells (KB, HeLa)^[1]
3.1.1 Adherent cells: No trypsin digestion required; use cells seeded on chamber coverslips.
3.2 Incubation conditions: Incubate live cells with 50 nM NRLD in pre-warmed HBSS at room temperature for 10 min.
3.3 Washing step: Wash the cells once with pre-warmed HBSS before imaging.

4. Control Setup
4.1 Set up a co-localization control using 500 nM BODIPY 493/503 (HY-W090090).
4.2 Positive controls are used to verify lipid droplet targeting specificity; blank controls are used to exclude fluorescent interference from reagents.

5. Detection and Analysis
5.1 Instrument: Confocal microscope.
5.1.1 Excitation wavelength: 488 nm; detect fluorescence in the 550-600 nm and 600-650 nm wavelength ranges, respectively.
5.2 Result analysis:
5.2.1 Changes in fluorescence intensity: A blue shift of the emission peak (higher I_550-600/I_600-650 ratio) indicates an environment with low polarity and tightly ordered lipid arrangement; a red shift of the emission peak (lower ratio) indicates an environment with higher polarity and disordered lipid arrangement.
5.2.2 Fluorescence localization: Specific localization to lipid droplets.
5.2.3 Color change: The pseudocolor ranges from blue-violet (low polarity, high lipid order) to orange-red (high polarity, low lipid order).

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