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.

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