9-7 Hongo 2-Chome, Bunkyo-Ku
Tokyo 113-0033, Japan

GolgiSeeingTM <Golgi Apparatus Green>
Catalog NO. FDV-0053
Research use only, not for human or animal therapeutic or diagnostic use.
This product has been commercialized under a license from the Nagoya Institute of Technology

Product Background
The Golgi apparatus is an organelle that plays various roles in physiological functions and is known as a centralcomponent of the protein secretory pathway. The Golgi apparatus has unique multiple-layered cisternal membrane
structures, with subdivided structures such as cis-Golgi, which is responsible for reciprocal vesicular transport withthe endoplasmic reticulum (ER), and trans-Golgi, which is the starting point of the secretory pathway. Since dynamic
morphological changes of the Golgi apparatus are essential for secretory function and dysfunction of the Golgi
apparatus has been implicated in a number of diseases, it is expected that the Golgi apparatus will be observed bylive cell imaging.
Two major methods have been used to stain the Golgi apparatus in living cells: The first is fluorescent staining
using fluorescently labeled ceramide derivatives (hereafter ceramide-FL). Ceramide lipids accumulate in the Golgi
apparatus during the metabolic pathway, and ceramide-FLs have been used to visualize the Golgi apparatus. However,
1) The Golgi selectivity of ceramide derivatives is low, 2) Ceramide derivatives also localize to the ER, etc., and 3)
Ceramide derivatives have high cytotoxicity and are quickly metabolized intracellularly. The second staining method
is to overexpress fluorescent proteins by fusing them to Golgi-specific expressed proteins (such as Giantin, Nacetylgalactosaminyltransferase, etc.). While this method allows visualization of the Golgi apparatus with a highdegree of specificity, it requires prior plasmid transfection, making it impossible to immediately observe the Golgi
apparatus when necessary. Furthermore, there are concerns overexpression of specific marker genes may affectphysiological functions of the Golgi apparatus. GolgiSeeingTM is a novel small molecule fluorescent reagent thatutilizes a Golgi apparatus selective localization motif discovered by Dr. Shinya Tsukiji's group at Nagoya Institute ofTechnology. Unlike conventional ceramide-FLs, it can stain the Golgi apparatus with a simple protocol that requires
only 10 minutes of addition to the culture medium. It also shows higher Golgi specificity than ceramide-FLs, allowing
Golgi apparatus-focused analysis (Figure 1). The GolgiSeeingTM can visualize the Golgi apparatus of target cells at
any desired timing without genetic manipulation and without bias to physiological functions caused byoverexpression, allowing the dynamic behavior of the Golgi apparatus to be observed under more physiologicalconditions. Under the non-wash protocol, GolgiSeeingTM visualizes not only the Golgi apparatus but also the plasma
membrane. This property allows simultaneous imaging of the Golgi apparatus and cell morphology (judged byplasma membrane).

Figure 1. Overview image of GolgiSeeingTM

GolgiSeeingTM (original compound name; mgc3MeFDA in Ref.1) has a unique structure consisting offluorescein diacetate (FDA) and N-myristoylated Gly-Cys dipeptide (mgc) linked by a linker, and three amide bondsin the mgc peptide chain are all methylated (Figure 2 left, upper). GolgiSeeingTM functions as a Golgi apparatusselective fluorescent probe through several processes in the cell. FDA is a modified fluorescein quenched to improvecell membrane permeability, and GolgiSeeingTM emits slight fluorescence before use. After GolgiSeeingTM penetrates
the cell membrane and enters the cell, acetyl groups are removed by endogenous esterases, and the green fluorescenceof fluorescein is restored (Figure 2 left, middle). The deacetylated form of GolgiSeeingTM transiently localizes mainly
to the ER and Golgi apparatus by the effect of N-myristoyl groups and is S-palmitoylation by endogenous Spalmitoylation enzymes. The Golgi apparatus selective localization motif is formed by adding palmitic acid to theCys side chain by enzymatic action (Figure 2 left, lower). Although the intrinsic palmitoylation modification isknown to localize to the Golgi apparatus and the plasma membrane, the effect of the three methyl groups biases theequilibrium toward the Golgi, which allows GolgiSeeingTM to selectively visualize the Golgi apparatus (Figure 2right).

Figure 2. Principle of GolgiSeeingTM
Table Live cell Golgi apparatus imaging methods
CategoryStaining
Golgi apparatus specificity

Genetically encoded
Fluorescent protein-fused Golgi
apparatus marker proteins
HighProtocolEasy(Exogenous gene transfection)Time> half day(time for protein expression)
High(Overexpression of exogenousproteins)

Influence on physiologicalfunction[ver. 2024/04]

Fluorescent probes
Fluorescent-labeled ceramide
derivatives (Ceramide-FL)
Low(High ER background)
Complicated(Require stepwise andtemperature-controlled protocol)>1 hourHigh(Exogenous ceramide’s toxicity)

GolgiSeeingTM
High(Low ER background)
Easy(Just addition to medium)10 minLow

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Description
Catalog Number: FDV-0053
Size: 0.1 mg
Molecular weight: 1258.5 g/mol
Solubility: Soluble in DMSO
Fluorescent characteristics:
Ex. 440-500 nm (maximum ~480 nm) / Em 500-560 nm (maximum ~520 nm)
Compatible with conventional FITC filter set*NOTE: GolgiSeeingTM has fluorescein diacetate (FDA), which is quenched by two acetates and emits aslight fluorescence. After hydrolysis of two acetic groups in cells by physiological esterases,
fluorescein is exposed and restores strong green fluorescence.

Reconstitution and Storage
Reconstitution: Stock solution recommended concentration 1 mM in 100% DMSO. Add 79 L of DMSO/vial toprepare 1 mM stock solution.
Storage (powder): Store powder at less than -20oC
Storage (solution): After reconstitution in DMSO, aliquot and store at less than -20°C.
Avoid repeated freeze-thaw cycles and recommend single use of each aliquot.
Important notice for stability
GolgiSeeingTM has a free-thiol group (See Figure 2 left), which is S-palmitoylated in cells and essential for
Golgi apparatus localization. This thiol group is easily oxidized to form a homodimer in physiological pH buffers,
and GolgiSeeingTM -dimer loses its Golgi apparatus localization function and may cause a background signal.
GolgiSeeingTM working solution (such as GolgiSeeingTM -containing medium or buffers) should be preparedjust before use. Importantly, GolgiSeeingTM may be gradually oxidized even in DMSO. After reconstitutionof GolgiSeeingTM in DMSO, use within one month to maintain good staining results.

How to use
General procedure for Golgi apparatus-selective staining in live cells*This procedure is an example of Golgi apparatus-selective staining
1. Prepare 10 M GolgiSeeingTM in serum-free medium such as DMEM just before use
NOTE: Empirically optimize and determine the concentration of GolgiSeeingTM for your experiments.
2. Remove the culture medium and wash cells with medium several times
3. Add GolgiSeeingTM -containing medium to cells
NOTE: Working solution of GolgiSeeingTM prepared in step-1 should be quickly used, as GolgiSeeingTM may
be oxidazed to form inactive dimer in medium.
4. Incubate cells at RT for 10 min
NOTE: Empirically optimize incubation time and temperature for your experiments.
5. Wash cells with 3 mg/ml BSA-containing medium over two times and add fresh serum-free and phenol redfree medium
NOTE: GolgiSeeingTM is a highly hydrophobic compound and non-specifically absorbed to plastic or glass
surface of imaging chambers. This non-specific absorption may cause the background signal offluorescent images. To reduce non-specific signals, we highly recommend using a wash buffer
containing BSA because BSA efficiently binds to excess GolgiSeeingTM and removes it from thesurface of imaging chambers. 3 mg/ml BSA-containing basal medium is the preferred choice ofGolgiSeeingTM -wash buffer.
6. Observe cells under live cell condition

General procedure for Golgi apparatus and plasma membrane staining in live cells*This procedure is an example of Golgi apparatus and plasma membrane staining
1. Prepare 2 M GolgiSeeingTM in serum-free and phenol red-free medium such as DMEM just before use
NOTE: Empirically optimize and determine the concentration of GolgiSeeingTM for your experiments.
2. Remove the culture medium and wash cells with medium several times
3. Add GolgiSeeingTM -containing medium to cells
NOTE: Working solution of GolgiSeeingTM prepared in step-1 should be quickly used, as GolgiSeeingTM may
be oxidazed to form inactive dimer in medium.
4. Incubate cells at RT for 10 min
NOTE: Empirically optimize incubation time temperature for your experiments.
6. Observe cells without wash-out step under live cell condition
Important Notice of Use1) GolgiSeeingTM will selectively localize to the Golgi apparatus through the S-palmitoylation on the free-thiolgroup by endogenous S-palmitoylation enzymes, as mentioned in Figure 2. Note that any drugs inhibiting Spalmitoylation may influence the Golgi apparatus-selective staining property of GolgiSeeingTM. Especially,
alkylation reagents of the free thiol (such as maleimide, iodoacetate, etc.) will critically inhibit S-palmitoylation
and are incompatible with GolgiSeeingTM.
2) GolgiSeeingTM has no fixable functional groups in the molecule and is not fixed by either paraformaldehyde(PFA) or methanol. GolgiSeeingTM is a specialized reagent for live cell imaging applications, not compatible
with fixed cell imaging and immunocytochemistry.
3) Long incubation time of GolgiSeeingTM may increase ER-derived fluorescent signal and reduce Golgi/ER ratio.
Conduct empirical optimization ranges for your experiments' incubation time and observation time course.

Reference data
Comparison between GolgiSeeingTM and a ceramide-based reagent
HeLa cells were treated with 10 M GolgiSeeingTM or 5 M ceramide-FL (as BSA complex). In the case ofGolgiSeeingTM, the protocol is a simple addition of GolgiSeeingTM into media final 10 M and incubated for 10 min.
After washing cells with 3 mg/ml BSA containing media, fluorescent images were captured by confocal laser
microscopy (Ex 488 nm/Em 500-600 nm). On the otherhand, ceramide-FL (BSA complex) -staining wasperformed by two protocols, simple addition orstepwise temperature-controlled protocol. The later
protocol cells were incubated with ceramide-FL (BSAcomplex) for 30 min at 4oC in HBSS, washed with icecold HBSS, and incubated in a fresh culture medium
for an additional 30 min at 37oC. Finally, cells were
washed with a fresh medium again and observed by
confocal laser microscopy. This stepwise protocol
requires over 1 hour. The ceramide-FL probe stains not
only the Golgi apparatus but also ER structure withhigh intensity non-specifically. GolgiSeeingTM wasable to visualize Golgi apparatus selectively andsuppressed non-specific ER staining.

[ver. 2024/04]

Download the latest datasheet from www.funakoshi.co.jp(Japanese)www.funakoshi.co.jp/exports (English)Organelle specificity
HeLa cells were co-stained with GolgiSeeingTM and organellemarkers. In the case of the Golgi apparatus marker,
overexpression of mCherry-Giantin fusion protein was used. In
the cases of ER, mitochondria, and lysosome, each organelle wasstained by organelle-specific chemical probes. The fluorescentsignal from GolgiSeeingTM is well corresponded with Golgimarker and weakly overlaps with ER. However, the signals are
not matched with mitochondria and lysosomes.

Effect of wash-out step on plasma membrane staining
HeLa cells were treated with 10 M GolgiSeeingTM for 10min and observed by confocal laser microscopy (Ex 488nm/Em 500-600 nm) with or without the wash step. For
wash-out, 3 mg/ml BSA-containing medium was used as
wash buffer. In the case of non-wash observation, thefluorescent signals of GolgiSeeingTM were observed in
not only the Golgi apparatus but also the plasma
membrane. As the Golgi apparatus is clearly distinguishedfrom the plasma membrane, non-wash staining is usefulfor simultaneous observation of the Golgi apparatus andcell morphology (the shape of the plasma membrane). The
wash-out step dramatically reduced the fluorescent signal
from the plasma membrane.

Various cell staining
Four types of cultured cells (HeLa, COS-7, HEK293,and Jurkat) were treated with 10 M GolgiSeeingTM for
10 min. After cell wash-out, cells were observed byconfocal laser microscopy (Ex 488 nm/ Em 500-600
nm). For all cells tested here, GolgiSeeingTM highly
selectively stained the Golgi apparatus.

Cellular toxicity
HeLa cells were seeded into a 12-well plate at 0.5 x 105
cells/well and cultured for 48 hours with/without 10 M
GolgiSeeingTM. After 2, 24, and 48 hours, cell numbers
were assessed by a cell counter. GolgiSeeingTM shows
little effect on cell proliferation.

Application data
Live cell time-lapse imaging on Brefeldin A-induced collapse of Golgi apparatus
HeLa cells were seeded on glass bottom dishes and treated with 10 M GolgiSeeingTM for 10 min. After washingcells with 3 mg/ml BSA-containing media, the cells were cultured in brefeldin A (final 1 M in 0.1% DMSO)containing medium or 0.1 % DMSO-containing medium as a negative control and observed under live cell condition
by confocal microscopy (Ex 488 nm/Em 500-600 nm). In the brefeldin A-treated cells, the fluorescent signal fromthe Golgi apparatus gradually disappeared.

Live cell time-lapse imaging during cell division
MDCK cells were seeded on a glass bottom dish, incubated with 2.5 M GolgiSeeingTM, and observed for 2 hours
by confocal laser microscopy (Ex 488 nm/ Em 500-600 nm) without washing step. As a non-wash procedure, thefluorescent signal of GolgiSeeingTM were observed not only in Golgi apparatus but also in plasma membrane. By
imaging of plasma mebrane structures, cell morphology was easily observed. According to the progression of cell
division, the Golgi apparatus disappeared (60 min) and was reconstituted in two daughter cells (90-120 min).

[ver. 2024/04]

Download the latest datasheet from www.funakoshi.co.jp(Japanese)www.funakoshi.co.jp/exports (English)Reference
1. Sawada et al.,ACS Chem. Biol., 18, 1047-1053 (2023) Palmitoylation-Dependent Small-Molecule Fluorescent
Probes for Live-Cell Golgi Imaging.

Disclaimer/免責事項
This product has been commercialized by Funakoshi Co., Ltd.
based on the results of academic research, and the advertisement
text, figures and manuals (hereinafter “Product information”) havebeen prepared based on published research reports on August,2023. The academic interpretation at the time of creation of theProduct Information may change in accordance with future
developments in the relevant research field and expansion ofvarious scientific findings, and the latest version and certainty of
the Product Information are not guaranteed. The specifications of
this product and the Product Information are subject to change
without notice. Please contact us for the latest information.
本商品は学術研究成果を基にフナコシ株式会社が商品化したもので、2023 年 8 月時点における公開研究報告を基に広告文章およびマニュアル（以下、商品資料）を作成しています。今後の当該研究分野の発展および各種学術知見の拡大にともない、商品資料作成時の学術的解釈が変更になる可能性があり、最新性・確実性を保証するものではありません。また、本商品の仕様および商品資料を予告なく変更する場合がございます。最新の情報に関しましては、弊社までご確認いただきますようお願い申し上げます。

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