Cell Navigator® NBD Ceramide Golgi Staining Kit *Green Fluorescence*

1. Treat cells as desired
2. Add C6 NBD Ceramide working solution and incubate at room temperature or 37°C for 15~30 minutes
3. Replace with the Staining Buffer and incubate at room temperature or 37°C for 15~30 minutes
4. Observe under microscope using FITC filter set
5. Optional: Fix cells with 4% Formaldehyde

Thaw one of each kit component 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) into the vial of C6 NBD Ceramide (Component A) and mix well.
Note         Store unused C6 NBD Ceramide stock solution at -20^o C in single use aliquotes. Avoid freeze-thaw cycles.

Add 10 µL of NBD Ceramide stock solution (100X) to 990 µL Staining Buffer (Component B) to make NBD Ceramide working solution.

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

1. Plate and treat cells as desired.

2. Add equal volume of C6 NBD Ceramide working solution directly in cell culture medium. Note: If you have cells in a 96 well plate with 100 µL/well cell culture medium then add 100 µL/well of C6 NBD Ceramide working solution.

3. Incubate at room temperature or 37 °C for 15~30min.
4. Remove the C6 NBD Ceramide working solution and replace with 100 µL/well of Staining Buffer (Component B).
5. Incubate at room temperature or 37 °C for 10~15 minutes.
6. Observe under a fluorescence microscope with FITC filter set.

Remove the staining buffer from Step 4, and add 100 µL/well/96-well plate of 4% formaldehyde fixative buffer (not supplied) to each well.
Note         For non-adherent cells, add desired amount (such as 2X10⁶ cells/mL) of 4% formaldehyde fixative buffer.

Incubate plates for 20 to 30 minutes at room temperature. Remove fixative. Wash the cells with PBS 2-3 times, and replace with 100 µL/well of Staining Buffer (Component B).

Note        The Cell Navigator™ NBD Ceramide Golgi Staining Kit also works well for aldehyde-fixed cells. After fixation, follow the staining protocol steps 2 to 6