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

<strong>The representative fluorescence image of GGR169 Ceramide Golgi Staining in HeLa cells.</strong> Cells were stained with 100 &micro;L of GGR169-ceramide working solution with Hoechst 33342 at 37 &deg;C for 20 minutes. An intensely fluorescent thread like structure, partially surround the nucleus, is identified as the Golgi apparatus.
<strong>The representative fluorescence image of GGR169 Ceramide Golgi Staining in HeLa cells.</strong> Cells were stained with 100 &micro;L of GGR169-ceramide working solution with Hoechst 33342 at 37 &deg;C for 20 minutes. An intensely fluorescent thread like structure, partially surround the nucleus, is identified as the Golgi apparatus.
<strong>The representative fluorescence image of GGR169 Ceramide Golgi Staining in HeLa cells.</strong> Cells were stained with 100 &micro;L of GGR169-ceramide working solution with Hoechst 33342 at 37 &deg;C for 20 minutes. An intensely fluorescent thread like structure, partially surround the nucleus, is identified as the Golgi apparatus.
<strong>The representative fluorescence images of GGR169 Ceramide Golgi Staining in HeLa cells in a pH dependent manner.</strong> HeLa cells were stained with 100 &micro;L of GGR169-ceramide working solution at 37 &deg;C for 20 minutes. Images were acquired using a fluorescence microscope equipped with Cy3 filter set &nbsp;before and after the addition of 30 mM of NH<sub>4</sub>Cl solution. A reduction in fluorescence intensity was observed after the addition of NH<sub>4</sub>Cl.
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Physical properties
SolventDMSO
Spectral properties
Excitation (nm)544
Emission (nm)570
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12171501
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OverviewpdfSDSpdfProtocol


See also: Golgi Apparatus
Excitation (nm)
544
Emission (nm)
570
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. Cell Navigator® TMR Ceramide Golgi Staining kit provides a simple and rapid way to stain Golgi in red fluorescence in live cells. Golgi apparatus is stained through the formation of the respective fluorescent metabolites. Cell Navigator® TMR Ceramide Golgi Staining Kit provides an optimized assay method for examining the morphology of the Golgi apparatus with a fluorescence microscope. In addition, GGR169-ceramide is pH-sensitive, thus uniquely serving as an excellent Golgi pH probe too.

Platform


Fluorescence microscope

ExcitationCy3/TRITC filter set
EmissionCy3/TRITC filter set
Recommended plateBlack wall/clear bottom

Components


Example protocol


AT A GLANCE

Protocol summary
  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 

Important
Thaw all the components 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.

GGR169-ceramide stock solution (100X)
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.

PREPARATION OF WORKING SOLUTION

GGR169-ceramide working solution
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.

SAMPLE EXPERIMENTAL PROTOCOL

Following protocol should be used for the guidelines and can be modified as per requirements.
  1. Plate and treat cells as desired.
  2. Add 100 µL of GGR169-ceramide working solution directly into cell culture medium.
  3. Incubate at room temperature or 37 °C for 15∼30 minutes.
  4. Remove the GGR169-ceramide working solution and wash once with DPBS or buffer of your choice.
  5. Add 100 µL/well of Staining Buffer (Component B).
  6. Observe under a fluorescence microscope with Cy3 filter set. 

Spectrum


Open in Advanced Spectrum Viewer
spectrum

Spectral properties

Excitation (nm)544
Emission (nm)570

Product Family


Images


References


View all 50 references: Citation Explorer
The role of ceramide in regulating endoplasmic reticulum function.
Authors: Zelnik, Iris D and Ventura, Ana E and Kim, Jiyoon L and Silva, Liana C and Futerman, Anthony H
Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids (2020): 158489
Chlamydia trachomatis-infected human cells convert ceramide to sphingomyelin without sphingomyelin synthases 1 and 2.
Authors: Tachida, Yuriko and Kumagai, Keigo and Sakai, Shota and Ando, Shuji and Yamaji, Toshiyuki and Hanada, Kentaro
Journal: FEBS letters (2020): 519-529
Structure, functions and regulation of CERT, a lipid-transfer protein for the delivery of ceramide at the ER-Golgi membrane contact sites.
Authors: Kumagai, Keigo and Hanada, Kentaro
Journal: FEBS letters (2019): 2366-2377
Neuronal ceroid lipofuscinosis related ER membrane protein CLN8 regulates PP2A activity and ceramide levels.
Authors: Adhikari, Babita and De Silva, Bhagya and Molina, Joshua A and Allen, Ashton and Peck, Sun H and Lee, Stella Y
Journal: Biochimica et biophysica acta. Molecular basis of disease (2019): 322-328
Both the N- and C- terminal regions of the Chlamydial inclusion protein D (IncD) are required for interaction with the pleckstrin homology domain of the ceramide transport protein CERT.
Authors: Kumagai, Keigo and Elwell, Cherilyn A and Ando, Shuji and Engel, Joanne N and Hanada, Kentaro
Journal: Biochemical and biophysical research communications (2018): 1070-1076
Activation of neutral sphingomyelinase 2 by starvation induces cell-protective autophagy via an increase in Golgi-localized ceramide.
Authors: Back, Moon Jung and Ha, Hae Chan and Fu, Zhicheng and Choi, Jong Min and Piao, Yongwei and Won, Jong Hoon and Jang, Ji Min and Shin, In Chul and Kim, Dae Kyong
Journal: Cell death & disease (2018): 670
Ceramide-transfer protein-mediated ceramide transfer is a structurally tunable flow-inducing mechanism with structural feed-forward loops.
Authors: Giordano, Giulia
Journal: Royal Society open science (2018): 180494
Antidepressants act by inducing autophagy controlled by sphingomyelin-ceramide.
Authors: Gulbins, Anne and Schumacher, Fabian and Becker, Katrin Anne and Wilker, Barbara and Soddemann, Matthias and Boldrin, Francesco and Müller, Christian P and Edwards, Michael J and Goodman, Michael and Caldwell, Charles C and Kleuser, Burkhard and Kornhuber, Johannes and Szabo, Ildiko and Gulbins, Erich
Journal: Molecular psychiatry (2018): 2324-2346
Ceramide Transporter CERT Is Involved in Muscle Insulin Signaling Defects Under Lipotoxic Conditions.
Authors: Bandet, Cécile L and Mahfouz, Rana and Véret, Julien and Sotiropoulos, Athanassia and Poirier, Maxime and Giussani, Paola and Campana, Mélanie and Philippe, Erwann and Blachnio-Zabielska, Agnieszka and Ballaire, Raphaëlle and Le Liepvre, Xavier and Bourron, Olivier and Berkeš, Dušan and Górski, Jan and Ferré, Pascal and Le Stunff, Hervé and Foufelle, Fabienne and Hajduch, Eric
Journal: Diabetes (2018): 1258-1271
Phosphoinositide binding by the PH domain in ceramide transfer protein (CERT) is inhibited by hyperphosphorylation of an adjacent serine-repeat motif.
Authors: Sugiki, Toshihiko and Egawa, Daichi and Kumagai, Keigo and Kojima, Chojiro and Fujiwara, Toshimichi and Takeuchi, Koh and Shimada, Ichio and Hanada, Kentaro and Takahashi, Hideo
Journal: The Journal of biological chemistry (2018): 11206-11217