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

The fluorescence image of NBD Ceramide Golgi Staining in HeLa cells. Cells were stained with 100 µL of C6 NBD Ceramide working solution at 37 °C for 20min and followed  with Hoechst 33342 stain. An intensely fluorescent threadlike structure, partially surround the nucleus, is identified as the Golgi apparatus.
The fluorescence image of NBD Ceramide Golgi Staining in HeLa cells. Cells were stained with 100 µL of C6 NBD Ceramide working solution at 37 °C for 20min and followed  with Hoechst 33342 stain. An intensely fluorescent threadlike structure, partially surround the nucleus, is identified as the Golgi apparatus.
The fluorescence image of NBD Ceramide Golgi Staining in HeLa cells. Cells were stained with 100 µL of C6 NBD Ceramide working solution at 37 °C for 20min and followed  with Hoechst 33342 stain. An intensely fluorescent threadlike structure, partially surround the nucleus, is identified as the Golgi apparatus.
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Spectral properties
Excitation (nm)467
Emission (nm)538
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
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OverviewpdfSDSpdfProtocol


Excitation (nm)
467
Emission (nm)
538
The Golgi apparatus is a complex of vesicles and folded membranes within the cytoplasm of most eukaryotic cells, involved in secretion and intracellular transport. It modifies proteins and lipids that have been built in the endoplasmic reticulum (ER) and prepares them for export outside of the cell. It also plays a significant role in the transport of lipids throughout the cell and the formation of lysosomes. This Cell Navigator® NBD Ceramide Golgi Staining kit provides a simple and rapid way to stain Golgi in live cells, or aldehyde-fixed cells selectively. C6 NBD Ceramide is administered to cells as a complex with bovine serum albumin (C6-NBD-Ceramide-BSA). Golgi apparatus is stained through the formation of the respective fluorescent metabolites. This Cell Navigator® NBD Ceramide Golgi Staining Kit provides an optimized assay method for examining the morphology of the Golgi apparatus with a fluorescence microscope.

Platform


Fluorescence microscope

Excitation488 nm
Emission525 nm
Recommended plateBlack wall/clear bottom
Instrument specification(s)FITC filterset

Components


Example protocol


AT A GLANCE

Protocol Summary
  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
Important Note

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

C6 NBD Ceramide stock solution (100X)

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

C6 NBD Ceramide 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

Staining protocol
  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.
Optional

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


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Spectral properties

Excitation (nm)467
Emission (nm)538

Product Family


Images


Citations


View all 44 citations: Citation Explorer
Molecular determinants of ER-Golgi contacts identified through a new FRET-FLIM system
Authors: Venditti, R., Rega, L. R., Masone, M. C., Santoro, M., Polishchuk, E., Sarnataro, D., Paladino, S., D&apos;Auria, S., Varriale, A., Olkkonen, V. M., Di Tullio, G., Polishchuk, R., De Matteis, M. A.
Journal: J Cell Biol (2019): 1055-1065
Functions of neutral ceramidase in the Golgi apparatus
Authors: Sakamoto, W., Coant, N., Canals, D., Obeid, L. M., Hannun, Y. A.
Journal: J Lipid Res (2018): 2116-2125
A Golgi Lipid Signaling Pathway Controls Apical Golgi Distribution and Cell Polarity during Neurogenesis
Authors: Xie, Z., Hur, S. K., Zhao, L., Abrams, C. S., Bankaitis, V. A.
Journal: Dev Cell (2018): 725-740 e4
Structure of the Golgi apparatus is not influenced by a GAG deletion mutation in the dystonia-associated gene Tor1a
Authors: Mitchell, S. B., Iwabuchi, S., Kawano, H., Yuen, T. M. T., Koh, J. Y., Ho, K. W. D., Harata, N. C.
Journal: PLoS One (2018): e0206123
Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide
Authors: Ishibashi, Y., Ito, M., Hirabayashi, Y.
Journal: Biochem Biophys Res Commun (2018): 1011-1018
A putative calcium-ATPase of the secretory pathway family may regulate calcium/manganese levels in the Golgi apparatus of Entamoeba histolytica
Authors: Rodriguez, M. A., Martinez-Higuera, A., Valle-Solis, M. I., Hern and es-Alej, undefined and ro, M., Chavez-Munguia, B., Figueroa-Gutierrez, A. H., Salas-Casas, A.
Journal: Parasitol Res (2018): 3381-3389
Comparison among different “revealers” in the study of accelerated blood clearance phenomenon
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
Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide
Authors: Back, M. J., Ha, H. C., Fu, Z., Choi, J. M., Piao, Y., Won, J. H., Jang, J. M., Shin, I. C., Kim, D. K.
Journal: Cell Death Dis (2018): 670
An inducible ER-Golgi tether facilitates ceramide transport to alleviate lipotoxicity
Authors: Liu, L. K., Choudhary, V., Toulmay, A., Prinz, W. A.
Journal: J Cell Biol (2017): 131-147
Sphingolipid metabolic flow controls phosphoinositide turnover at the trans-Golgi network
Authors: Capasso, S., Sticco, L., Rizzo, R., Pirozzi, M., Russo, D., Dathan, N. A., Campelo, F., van Galen, J., Holtta-Vuori, M., Turacchio, G., Hausser, A., Malhotra, V., Riezman, I., Riezman, H., Ikonen, E., Luberto, C., Parashuraman, S., Luini, A., D&apos;Angelo, G.
Journal: EMBO J (2017): 1736-1754