NRERf

Guide
(The following is our recommended protocol. This protocol is for guidance only and should be modified according to your specific needs).

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

2. Working Solution Preparation
2.1 Diluent: Warm HBSS.
2.2 Working concentration: 200 nM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type: Adherent cells (KB, HeLa)^[1].
3.1.1 Adherent cells: No trypsinization mentioned; cells are seeded onto chambered coverglass 24 h before imaging.
3.2 Incubation conditions: Incubate cells with 200 nM NRERf working solution for 20 min at 37°C.
3.3 Washing steps: Wash cells once with warm HBSS before imaging.

4. Controls
4.1 Colocalization control: Co-incubate cells with 200 nM ERTracker Green (Invitrogen) alongside NRERf to validate endoplasmic reticulum targeting.

5. Detection & Analysis
5.1 Instrument: Confocal fluorescence microscope.
5.1.1 Excitation wavelength: 488 nm; emission detected at 550–600 nm and 600–650 nm.
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: Ratiometric analysis of I₅₅₀₋₆₀₀/I₆₀₀₋₆₅₀ reflects local membrane polarity; higher ratio indicates lower polarity (higher lipid order).
5.2.2 Fluorescence localization: Selectively targets endoplasmic reticulum, showing high colocalization with ERTracker Green.
5.2.3 Color changes: Red fluorescence emission; pseudocolor ratiometric images show orange tones for the endoplasmic reticulum, indicating a relatively polar (lower lipid order) microenvironment.

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