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Cell Navigator® NBD Ceramide Golgi Staining Kit *Green Fluorescence*
Example protocol
AT A GLANCE
- Treat cells as desired
- Add C6 NBD Ceramide working solution and incubate at room temperature or 37°C for 15~30 minutes
- Replace with the Staining Buffer and incubate at room temperature or 37°C for 15~30 minutes
- Observe under microscope using FITC filter set
- Optional: Fix cells with 4% Formaldehyde
Thaw one of each kit component at room temperature before starting the experiment.
CELL PREPARATION
For guidelines on cell sample preparation, please visit https://www.aatbio.com/resources/guides/cell-sample-preparation.html
PREPARATION OF STOCK SOLUTIONS
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 -20o C in single use aliquotes. Avoid freeze-thaw cycles.
PREPARATION OF WORKING SOLUTION
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.
SAMPLE EXPERIMENTAL PROTOCOL
- Plate and treat cells as desired.
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.
- Incubate at room temperature or 37 °C for 15~30min.
- Remove the C6 NBD Ceramide working solution and replace with 100 µL/well of Staining Buffer (Component B).
- Incubate at room temperature or 37 °C for 10~15 minutes.
- 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
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| Cell Navigator® TMR Ceramide Golgi Staining Kit *Red Fluorescence* | 544 | 570 |
Citations
Authors: Smith, Danielle M and Liu, Brian Y and Wolfgang, Michael J
Journal: Nature Communications (2024): 4469
Authors: Venditti, R., Rega, L. R., Masone, M. C., Santoro, M., Polishchuk, E., Sarnataro, D., Paladino, S., D'Auria, S., Varriale, A., Olkkonen, V. M., Di Tullio, G., Polishchuk, R., De Matteis, M. A.
Journal: J Cell Biol (2019): 1055-1065
Authors: Liang, Kaifan and Wang, Lirong and Su, Yuqing and Liu, Mengyang and Feng, Rui and Song, Yanzhi and Deng, Yihui
Journal: European Journal of Pharmaceutical Sciences (2018): 210--216
Authors: Sakamoto, W., Coant, N., Canals, D., Obeid, L. M., Hannun, Y. A.
Journal: J Lipid Res (2018): 2116-2125
Authors: Xie, Z., Hur, S. K., Zhao, L., Abrams, C. S., Bankaitis, V. A.
Journal: Dev Cell (2018): 725-740 e4
Certificate of origin