Cell Navigator® TMR Ceramide Golgi Staining Kit *Red Fluorescence*

1. Treat cells as desired
2. Add GGR169-ceramide working solution and incubate at room temperature or 37 °C for 15∼30 minutes
3. Replace with the Staining Buffer
4. Observe under microscope using Cy3 filter set

Thaw all the components at room temperature before starting the experiment.

For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles

Add 100 µL of DMSO (Component C) to GGR-ceramide (Component A) to make GGR169-ceramide stock solution (100X).
Note        Store the unused GGR-ceramide stock solution at -20 °C in single use aliquots to avoid freeze thaw cycles.

Add 10 µL of GGR169-ceramide stock solution (100X) to 990 µL of Staining Buffer (Component B) to make GGR169-ceramide working solution.

Optional: Add 10 µL of Hoechst 33342 (Component D) to 1 mL GGR169-ceramide working solution for nuclear stain. Observe under fluorescence microscope with DAPI filter set.

Following protocol should be used for the guidelines and can be modified as per requirements.

1. Plate and treat cells as desired.

2. Remove the cell culture medium. Optional: Cells can be washed with buffer of your choice.

3. Add 100 µL of GGR169-ceramide working solution directly into cell culture medium.

4. Incubate at room temperature or 37 °C for 15∼30 minutes.

5. Remove the GGR169-ceramide working solution and wash once with DPBS or buffer of your choice.

6. Add 100 µL/well of Staining Buffer (Component B).

7. Observe under a fluorescence microscope with Cy3 filter set.