NRLyso

Guidelines (The following is a recommended experimental protocol for guidance only, and should be adjusted according to your specific requirements)

1. Stock Solution Preparation
1.1 Concentration recommendation: 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 description: Adherent cells (KB, HeLa cells)^[1]:
3.1.1 Adherent cells: No trypsin digestion required; seed cells onto chamber coverslips (seed 24 h before microscopic imaging for optimal imaging results).
3.2 Incubation conditions: Incubate with 50 nM NRLyso at 37°C for 45 min.
3.3 Washing step: Wash cells once with pre-warmed HBSS before imaging.

4. Control Setup
4.1 Use 50 nM LysoTracker Green DND-26 (HY-D1296) to set up a colocalization control for verifying lysosome targeting specificity.
4.2 Set up a blank control to eliminate fluorescence interference from the reagent itself.

5. Detection and Analysis
5.1 Instrument type: Confocal fluorescence microscope.
5.2 Excitation/emission wavelengths: Excitation wavelength is 488 nm; detect emission light in the 550-600 nm and 600-650 nm wavelength ranges, respectively.
5.3 Result analysis:
5.3.1 Fluorescence localization: Selectively localizes to lysosomes in living cells.
5.3.2 Color shift: The emission peak undergoes a blue shift in environments with low polarity and high lipid order, and a red shift in environments with high polarity and low lipid order.
5.3.3 Fluorescence intensity change: The fluorescence quantum yield of NRLyso in ordered lipid phases is higher than that in disordered lipid phases or buffer. The fluorescence intensity ratio at 550-600 nm and 600-650 nm can be calculated; a higher ratio indicates lower local polarity and higher lipid order, while a lower ratio indicates higher polarity and lower lipid order.
5.3.4 Calculation of lipid order parameter (LOP): Convert ratio data to LOP via calibration with large unilamellar vesicles (LUVs) of DOPC (Ld phase) and SM/Chol (Lo phase); the LOP value of lysosomes is lower than that of cell membranes and mitochondria.

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