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

NucleoSeeingTM <Live Nucleus Green>
Catalog NO. FDV-0029
Research use only, not for human or animal therapeutic or diagnostic use.

Product Background
Cell nucleus is one of the most important organelle which stores DNA and chromatin complex in eukaryotic
cells and play important roles in various biological functions such as gene expression and epigenetic modulation. To
monitor dynamics of cell nucleus in live cells, many cell nucleus-specific dyes have been developed. Hoechst33342
dye is one of the most famous dyes and is used for various applications because of its membrane permeable property.
However, Hoechst33342 requires UV excitation (~350 nm) and has blue fluorescent property (~450 nm). As UV
excitation shows strong photo-toxicity for cells, Hoechst33342 is not suitable for monitoring nucleus dynamics onlive cells. Several visible-light excitable dyes for nucleus imaging on live cells had been commercially available,
however, they have some disadvantages such as DNA/RNA specificity, or cell toxicity.
NucleoSeeingTM is a novel type of nucleus imaging probe in live cells (Ref.1). This probe has a unique chemical
structure containing diacetyl-fluorescein and Hoechst33342 as DNA-binding tag linked by the optimal PEG linker.
Although NucleoSeeingTM has two fluorophores, fluorescence of this probe is strongly quenched under no DNA
conditions. Only in the presence of double stranded DNAs, it binds to DNA and emit fluorescence excited at 488 nm
and detected at 520 nm (Figure 1). Based on this photoproperty, NucleoSeeingTM allows to monitor cell nucleus withhighly S/N value against cytosol or other organelles. Furthermore, this probe specifically interacts with DNA sameas Hoechst dyes. Compared with Hoechst33342, NucleoSeeingTM has advantages which are not only lower phototoxicity, but also lower compound’s cellular toxicity. Staining of various mammalian cultured cell lines and cultured
mouse slice brain tissue were validated. Owing to its low photo- and cytotoxicity, long-term time lapse imaging (~20hours) of nucleus and chromatin dynamics is well succeeded. Not only mammalian cells, but also some plant cells
were tested, such as Arabidopsis thaliana Guard cells and epidermal cells (Ref.2). Although plant cells show
autofluorescence derived from chloroplasts, NucleoSeeingTM could separate nucleus signal from autofluorescencesignal from chloroplasts.
Inadditiontonucleus-specificimaging,

NucleoSeeingTM can be applied for nucleus-specific pH
sensing. A principle is based on pH-dependent
fluorescent properties of fluorescein and Hoechst33342,
components of NucleoSeeingTM. When this probe isexcited at 405 nm, values of fluorescent ratio(FFluorescein/FHoechst) depend on pH during pH5.5-8.5. This
product is world-first commercially available nucleusspecific pH sensing reagent. Please find the “Appendix”
for pH sensing.
Figure 1. Overview of NucleoSeeingTM

Description
Catalog Number : FDV-0029
Size : 0.1 mg
Molecular weight : 1425 g/mol as tri-TFA salt
Solubility : Soluble in DMSO
Fluorophore : Fluorescein (green fluorescent dye)
Ex/Em: 488 nm/520 nm*FITC filter sets are available.

Application- Live cell nucleus imaging of cultured cells, cultured tissues- Live cell nucleus imaging of plant cells- Cell nucleus staining of fixed cells- Cell nucleus-specific pH sensing (See “Appendix”)

Reconstitution and Storage
Reconstitution : stock solution in 100% DMSO.
Storage (solution) :Store powder at -20oC.
After reconstitution in DMSO, aliquot and store at -20 °C. Avoid repeated freeze-thaw cycles.
Protect from light.

How to use
General procedure of nucleus imaging
1. Prepare 1-20 M NucleoSeeingTM in fresh medium
NOTE: Please optimize concentration of reagent for your experiments. NucleoSeeingTM is compatible with
FBS-containing medium. When the user NucleoSeeingTM in FBS-containing medium, relatively higher
concentration (>5 M) of NucleoSeeingTM is recommended. Detail is described in “Application data”.
2. Remove culture medium
3. Add NucleoSeeingTM -containing medium to cells
4. Incubate cells at 37oC for over 15 min
5. Wash cells by PBS or medium (Option)
NOTE: For short-term observation, reversible staining or high sensitive detection, wash out is recommended.
On the other hand, for long-term observation, no wash out/medium change is highly recommended.
6. Observe cells under live condition or after fixation by 4% PFA and methanol[ver. 2024/05]

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Application data
Basic information of NucleoSeeingTM
Left: Fluorescent spectrum excited at 460 nm in the presence and absence of synthetic DNA. NucleoSeeingTM shows
strong green emission only in the presence of DNA.
Light : Fluorescent imaging by conventional Hoechst33342 and NucleoSeeingTM. NucleoSeeingTM is well overlappedwith DNA-specific dye Hoechst33342.

Cell toxicity
Cell toxicities of Hoechst33342 and NucleoSeeingTM were tested by the MTT assay.
While Hoechst shows potent cell toxicity dose-dependent manner, NucleoSeeingTM
keeps cell viability at least 5 M.

Reversible staining of NucleoSeeingTM
After HeLa cells were treated 1 M
Hoechst33342 and NucleoSeeingTM
for 15 min, cells were washed by
PBS and cultured for 24 hours.
While over 80% of blue signal of
Hoechst33342 was still remained,
green signals of NucleoSeeingTM
were dramatically reduced within
12 hours. NucleoSeeingTM can beused for reversible nucleus staining.
Influence of FBS on NucleoSeeingTM staining
To check any influence of FBS on nucleus staining by
NucleoSeeingTM, HeLa cells were treated 1 M
NucleoSeeingTM in serum-free medium or 1, 5, 10 M
NucleoSeeingTM in 10% FBS-containing media for 15
min. Images were acquired by confocal microscopy (Ex.
488/ Em. 500-600 nm) in the same condition (laser powerand detection sensitivity). Compared with 1 M in serumfree condition, nucleus fluorescent intensity stained by 1
M with 10% FBS was partially reduced. Higherconcentration such as 5 M or 10 M of NucleoSeeingTM
in FBS condition recovered fluorescent intensity.

Staining of various mammalian cells
Six types of mammalian cell lines were treated with 1 M
NucleoSeeingTM for 15 min and observed in live cell condition.

Staining of cultured slice tissue
Cultured mouse hippocampal brain tissue was treated with 20 M
NucleoSeeingTM for 15 min and observed in non-fixed condition.

Long-term time lapse imaging of mitotic cells
HeLa cells on the glass bottom dish were treated with 0.5 M NucleoSeeingTM in 10% FBS-containing DMEM for
1.5 hours. After treatment of NucleoSeeingTM, cells were observed for 20 hours by confocal microscopy without
any washout or medium change (Time lapse condition: Ex 488/Em 500-600 nm, 60x oil lens, 10 min interval).
Several mitotic cells were observed (white and orange arrow indicated cells in the mitotic process).
*Movie data is available on our website.

[ver. 2024/05]

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Staining of fixed cells
Left: Staining after fixation. PFA-fixed HeLa cells were treated by 1 M NucleoSeeingTM for 15 min, washed by
PBS and observed (Ex. 488 nm/ Em 520 nm).
Right: Fixation after staining. HeLa cells were treated by 5 M NucleoSeeingTM for 15 min in live cell condition.
After wash the cells, the cells were fixed with 4% PFA. The cells were observed (Ex. 488 nm/ Em 520 nm).
NOTE: MeOH fixation is also validated but signal may be decreased before MeOH fixation.

Staining of Arabidopsis thaliana Guard cells
Arabidopsis thaliana leaves were treated with 20 M NucleoSeeingTM in 10 mM MES-KOH, 50 mM KCl (pH 6.2)
buffer for 60 min and observed in living condition (Ex. 488 nm and Em. 490-555 nm for NucleoSeeingTM, Em. >615
nm for chloroplast as autofluorescence). Green signals from NucleoSeeingTM were clearly separated from plant
autofluorescence derived from chloroplast.

Staining of Arabidopsis thaliana epidermal cells
Arabidopsis thaliana leaves were treated with 20 M NucleoSeeingTM in 10 mM MES-KOH, 50 mM KCl (pH 6.2)
buffer for 60 min and observed in living condition. Ex. 488 nm and Em. 490-555 nm for NucleoSeeingTM.

Appendix : Nucleus-specific pH sensing
Ref.3 showed NucleoSeeingTM can be used in nucleus-specific pH sensing in live cells. As cell nucleus isseparated from cytosol by nuclear membrane with unique double lipid-bilayers, it is considered that the physicalenvironment of nucleus is different from cytosol. Some evidences suggested that nuclear-specific proton pumps (H+ATPases) regulate intra-nuclear pH condition and may play important roles in nuclear physiology. However, researchtools for monitoring nucleus pH sensing are limited.
Nucleus imaging by NucleoSeeingTM, Ex. 488 nm/ Em. 520 nm is comfortable. In the case of nucleus-specific
pH sensing, excitation at 405 nm and double detection at 520 nm and at 460 nm should be used. The ratio offluorescent intensity (F520/F460) depends on pH (Figure A1). To monitor nucleus pH, cell-based control experimentswith different pH buffer containing nigericin, a representative K+/H+-ionophore, required (Figure A2). Calibration
curves should be obtained every experiments.

Figure A1 pH-dependent fluorescent profile
Left: Fluorescence spectral changes of synthetic intracellular form of NucleoSeeingTM (see “NOTE”below) with synthetic DNA upon pH change measured in 50 mM MOPS, 100 mM NaCl. Excitation at
405 nm.
Right: pH-dependent changes in the fluorescence intensity at 460 nm (F460) and 520 nm (F520), and theratio (F520/F460)
NOTE : NucleoSeeingTM containing hoechst33342-diacetylfluorescein is highly optimized for live cellimaging. It enters into cells and is rapidly deacetylated to hoechst33342-fluorescein byintracellular esterases. So, the deacetyl-form of NucleoSeeingTM is active probe for DNA staining.
Figure A1 was obtained from “chemically synthesized deacetyl-form of NucleoSeeingTM”.
Please note this data is not reproduced by using our NucleoSeeingTM itself in vitro.

Additionally required reagents- nigericin (a representative K+/H+ ionophore)- pH standard buffers (ex. 10 mM MOPS, 130 mM KCl, 10 mM NaCl, 1 mM MgSO4) with pH range 5.5-8.5[ver. 2024/05]

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How to use
1. Prepare pH-standard buffers for calibration curve (10 mM MOPS, 130 mM KCl, 10 mM NaCl, 1 mM MgSO4)
at pH 5.5-8.5 and 10 g/ml nigericin -containing pH-standard buffers.
2. Cells were incubated with NucleoSeeingTM (1-10 M) for 15 min
3. After wash cells twice with pH-standard buffers of the indicated pH, the cells were incubated with 10 g/ml
nigericin -containing pH-standard buffers at 37oC for 30 min
4. The cells were observed at Ex 405nm and Em 520 nm and 460 nm.
Data example

Figure A2 in cellulo ratiometric imaging
Cells were incubated with 5 M NucleoSeeingTM for 15 min, washed and observed after nigericintreatment at the indicated pH or directly as intact cells. Excitation at 405 nm and detection at 430-510 nm
as Hoechst filter and 520-620 nm as Fluorescein filter.

Reference
1. Nakamura et al., Chem. Commun., 50, 6149-6152 (2014) Hoechst tagging: a modular strategy to design syntheticfluorescent probes for live-cell nucleus imaging.
2. Ueda et al., ACS Cent. Sci., 3, 462-472 (2017) Noncanonical function of a small-molecular virulence factor
coronatine against plant immunity: an in vivo raman imaging approach.
3. Nakamura and Tsukiji, Bioorg. Med. Chem. Lett., 27, 3127-3130 (2017) Ratiometric fluorescence imaging ofnuclear pH in living cells using Hoechst-tagged fluorescein.

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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 January,
2019. 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、2019 年 1 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。

[ver. 2024/05]

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