ER Tracker Yellow

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 Solvent: DMSO.
1.2 Concentration recommendation: 1 mM.

2. Working Solution Preparation
2.1 Diluent: Pre-warmed cell culture medium.
2.2 Working concentration: 0.25 μM.
2.3 Note: Adjust working solution concentration as needed; prepare fresh before use.

3. Staining Procedure
3.1 Sample type:[1]
3.1.1 Adherent cells (HeLa, NIH3T3, Chang liver, C2C12 myotubes). No trypsinization specified for staining.
3.1.2 Primary cells (brown adipocytes).
3.2 Incubation conditions: Incubate cells with 0.25 μM ER thermo yellow working solution at 37°C, 5% CO₂ for 30 minutes.
3.3 Washing steps:
3.3.1 Live cells: Wash once with fresh pre-warmed culture medium.
3.3.2 Fixed cells: After staining, fix with 4% formaldehyde solution for 30 minutes at room temperature, then wash three times with 20 mM HEPES buffer solution (pH 5.0, 6.0, 7.0, or 8.0).

4. Controls
4.1 Set up DMSO control for ionomycin-induced heat production experiments.
4.2 Use commercially available ER tracker as a temperature-insensitive control for staining and temperature sensitivity validation.

5. Detection & Analysis
5.1 Instrument: Confocal laser scanning microscope.
5.1.1 Ex/Em wavelength: 559/581 nm; use Texas red (543 nm laser) filter set for imaging.
5.2 Result analysis:
5.2.1 Fluorescence intensity changes: Fluorescence intensity decreases with increasing temperature, showing a 3.9%/°C sensitivity in live and fixed HeLa cells; returns to baseline after cooling. Fluorescence intensity decreases upon ionomycin-induced heat production at the ER, then recovers to initial levels.
5.2.2 Fluorescence localization: Selectively localizes to the endoplasmic reticulum, confirmed by co-localization with ER tracker green (Pearson’s correlation coefficient = 0.90).
5.2.3 Color changes: Red fluorescence.

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