NRERcl

Guidelines (The following recommended experimental protocol is for guidance only and should be adjusted according to your specific requirements)
1. Stock Solution Preparation
1.1 Solvent: Dry DMF.
1.2 Concentration Recommendation: It is generally recommended to prepare a high-concentration stock solution at 1-10 mM.
2. Working Solution Preparation
2.1 Diluent: Pre-warmed Hanks' Balanced Salt Solution (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 No trypsin digestion required; stain cells directly after seeding onto chambered coverslips.
3.2 Incubation Conditions: Incubate with 50 nM NRERCl working solution at 37°C for 20 min.
3.3 Washing Step: Wash cells once with pre-warmed HBSS before imaging.
4. Control Setup
4.1 Colocalization Control: Co-incubate with 500 nM ERTracker Green (HY-D1297) (a reference marker for endoplasmic reticulum).
4.2 Set up negative controls, positive controls and blank controls.
4.3 Positive controls are used to validate the experimental system; blank controls are used to exclude fluorescent interference from reagents.
5. Detection and Analysis
5.1 Instrument Type: Laser scanning confocal microscope.
5.2 Excitation/Emission Wavelengths: Excitation wavelength: 488 nm; detect emission wavelengths at 550-600 nm and 600-650 nm.
5.3 Result Analysis:
5.3.1 Fluorescence Intensity Change: Ratio analysis of I_550-600/I_600-650 can reveal changes in local polarity and lipid order.
5.3.2 Fluorescence Localization: Selectively localizes to the endoplasmic reticulum (ER).
5.3.3 Color Change: Emits red fluorescence; a blue shift of the emission peak indicates a more ordered (less polar) lipid environment, while a red shift of the emission peak indicates a more disordered (more polar) lipid environment.
5.3.4 Lipid order parameters (LOP) can be calculated using calibration data from model lipid vesicles (DOPC LUVs for the disordered phase, SM/Chol LUVs for the ordered phase) to quantitatively analyze lipid order in the endoplasmic reticulum.