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DNA-specific Cell Nucleus Live Imaging Reagent NucleoSeeing(Live Nucleus Green)

Date:March 12 2019Web Page No:81204

Funakoshi Co.,Ltd.

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NucleoSeeing is a nuclear staining reagent for live cell imaging that binds specifically to DNA and emits green fluorescence. It exhibits a high S/N values not only in animal cells and tissues, but also in Arabidopsis thaliana leaf cells, making it excellent for observing nuclear dynamics in live cells. NucleoSeeing can also be applied for nucleus-specific pH sensing.

Example of use, NucleoSeeing<sup>™</sup>

Staining Examples of using NucleoSeeing

HeLa cell, Arabidopsis thaliana leaf tissue and Mouse brain pippocampus were observed by live cell imaging, respectively; see each data for details.

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Cell Nuclear Imaging in Living Cells

Cell nucleus is one of the most important organelle, serving a DNA storehouse and responsible for cell division and epigenetic modulation. Therefore, monitoring nucleus-dynamics in live cells is considered a very important issue. Many cell nucleus-specific dyes have been developed. While nucleic acid-responsive blue fluorescent dyes, such as Hoechst series and DAPI have been widely used, these blue fluorescent dyes require UV excitation, which shows strong photo-toxicity for cells and not applicable to live cell imaging. Recently, several green or red fluorochromes for nucleus imaging on live cells have been commercially available, however, they have some disadvantages such as DNA/RNA specificity, or cell toxicity.

NucleoSeeing is a DNA-responsive probe that has a DNA-specific binding tag and emits green fluorescence in the presence of DNA, enabling live cell imaging in cultured cells, cultured tissues, and plant cells such as Arabidopsis thaliana leaf cells. The product exhibits low cytotoxicity, high DNA-specific S/N value, and excellent cell nuclear staining. Fixed cells can also be observed, allowing for flexible planning of applications.
Furthermore, it has unique pH-dependent fluorescence properties and can be applied for nucleus-specific pH sensing. With the recent discussion on the importance of nuclear pH, it is expected to be used as a dedicated reagent for nuclear pH measurement.

For more information on nuclear pH measurement, please click here.

Comparison of nuclear staining reagents

Reagent Name Optical Properties Physical Properties Observation Method
Wavelength
Ex/Em(nm)
Fluorescence Photo-toxicity Nucleus Specificity Cell Membrane Permeability Cyto-toxicity Live Cell Fixed Cell
NucleoSeeing 488 / 520 Green Low High Permeable Low Good Good
Hoechst 350 / 461 Blue High High Permeable High Good Good
DAPI 350 / 461 Blue High High Impermeable Unknown No Good
Company X 485 / 498 Green Low Middle Permeable Unknown Good Good
Company Y 646 / 680 Red Low High Permeable High Good Good

* Photo-toxicity: cytotoxicity due to light irradiation

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Principle

NucleoSeeing is composed of green fluorescent dye and DNA binding tag. Under DNA free condition, NucleoSeeing is in folded conformation and no fluorescent is emitted. However, once it binds to DNA, the conformation will change and emitts green fluorescence.

Schematic diagram of NucleoSeeing

Schematic diagram of NucleoSeeing

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Features

  • High S/N ratio
  • Excitation / Emission : 488 nm / 520 nm
  • Low cytotoxicity
  • Can be used for both live and fixed cells
  • Can be used for animal samples and plant cells.
    (validated in leaf and guard cell of A. thaliana)
  • Reversible : Can be washed out by replacing the medium
  • Can be used as nucleus pH sensor

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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 (pH 6-8)

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Example Data

Index

DNA-responsive fluorescent properties and nuclear-specific staining

Nuclear-specific staining
  • Left: NucleoSeeing shows strong green fluorescent only in the presence of DNA. Excitation 488 nm.
  • Right: Co-staining with DNA-specific dye Hoechst33342 (Blue) and NucleoSeeing(Green) in living cells. They overlap very well.

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Cell toxicity

Cell toxicities of Hoechst33342, widely used as blue nuclear staining reagent, and NucleoSeeing were tested by MTT assay. While Hoechst causes cell death at 5 μM, NucleoSeeing keeps cell viability at least 5 μM.

Cytotoxicity of NucleoSeeing

Cell toxicity of NucleoSeeing

Reversible staining

To evaluate the cellular retention, cells were treated with 1 μM Hoechst33342 and NucleoSeeing for 15 min. The medium was changed to remove excess reagents, and fluorescence intensity was observed for 24 hours. While over 80% of Hoechst33342 signal was still remained, NucleoSeeing signals were dramatically reduced within 12 hours. NucleoSeeing can be used for reversible nucleus staining.

Reversible staining of NucleoSeeing

Reversible staining of NucleoSeeing

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Staining of various cultured cells

Various cell lines were treated with 1 μM NucleoSeeing for 15 min. After medium change, cells were observed in live cell condition. Nucleus-specific signals were observed in each cell lines.

Various cultured cells were stained with NucleoSeeing

Various cultured cells were stained with NucleoSeeing

Long-term time lapse imaging

HeLa cells on a glass bottom dish were treated with 0.5 μM NucleoSeeing in DMEM (+10% FBS) for 1.5 hours. The 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). The process of mitotic cell division and cell proliferation was observed.

If you cannot play the video, please see here.

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Staining of mouse brain (hippocampus) slice culture tissue

Cultured mouse hippocampal brain tissue was treated with 20 μM NucleoSeeing for 15 min and observed in non-fixed condition.

Mouse brain (hippocampus) slice culture tissue was stained with NucleoSeeing

Mouse brain (hippocampus) slice culture tissue was stained with NucleoSeeing

Influence of FBS

To check any influence of FBS, cells were treated NucleoSeeing 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 power and detection sensitivity). Compared with 1 μM NucleoSeeing in serum-free condition, nucleus fluorescent intensity stained by 1 μM with 10% FBS was partially reduced. Higher concentration of NucleoSeeing in FBS condition recovered fluorescent intensity.
NOTE: NucleoSeeing is compatible with FBS-containing medium, however the staining property is lower than FBS-free condition. It is recommended to optimize the reagent concentration.

Influence of FBS on NucleoSeeing staining

Influence of FBS on NucleoSeeing staining

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Staining of fixed cells

Fixed cells (HeLa cells) were stained with NucleoSeeing

Fixed cells (HeLa cells) were stained with NucleoSeeing

  • Left: PFA-fixed HeLa cells were treated by 1 μM NucleoSeeing for 15 min, washed by PBS and observed.
  • Right: HeLa cells were treated by 5 μM NucleoSeeing for 15 min in live cell condition. After wash the cells, the cells were fixed with 4% PFA and observed.
  • NOTE: Methanol fixation is also validated but signal may be decreased.

Staining of Arabidopsis thaliana Guard cells

Leaf slices of Arabidopsis thaliana were treated with 20 μM NucleoSeeing for 60 min. After washing, the slices were observed in living condition. When excited at 488 nm, nucleus of guard cells at closed state were observed in green fluorescence (Em. 490-555 nm), and autofluorescence derived from chloroplasts was observed (Em. >615 nm). Using this reagent, cell nucleus can be observed separately from autofluorescence of chloroplasts, and it is expected as a nuclear dynamics imaging reagent for plant cells.

After finding nuclear staining in Arabidopsis thaliana Guard cells in original paper 2, it was reported in original paper 4 that guard cells are specifically stained when stomata is closed and de-stained when stomata open. Using this phenomenon, a simple and high-throughput evaluation method for stomatal opening and closure using this reagent has been proposed. For details, please refer to original article 4.

<i>Arabidopsis thaliana</i> Guard cells were stained with NucleoSeeing<sup>™</sup>

Arabidopsis thaliana Guard cells were stained with NucleoSeeing

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Staining of Arabidopsis thaliana epidermal cells

Arabidopsis thaliana leaves were treated with 20 μM NucleoSeeing for 60 min and observed in living condition. Nucleus-specific staining was confirmed in epidermal cells.

<i>Arabidopsis thaliana</i> epidermal cells were stained with  NucleoSeeing<sup>™</sup>

Arabidopsis thaliana epidermal cells were stained with NucleoSeeing

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Original Papers

This product, NucleoSeeing is equal to hoeAc2FL in the papers below.

  1. Nakamura, A., et al., "Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging.", Chem. Commun., 50, 6149~6152 (2014). [PMID:24776726]
  2. Ueda, M., et al., "Noncanonical function of a small-molecular virulence factor coronatine against plant immunity: an in vivo raman imaging approach."ACS Cent. Sci., 3 (5), 462~472 (2017). [PMID:28573209]
  3. Nakamura, A. and Tsukiji, S., "Ratiometric fluorescence imaging of nuclear pH in living cells using hoechst-tagged fluorescein.", Bioorg. Med. Chem. Lett., 27 (14), 3127~3130 (2017). [PMID:28558968]
  4. Takaoka, Y., et al., "Hoechst-tagged Fluorescein Diacetate for the Fluorescence Imaging-based Assessment of Stomatal Dynamics in Arabidopsis thaliana.", Sci. Rep., 10, 5333 (2020). (
  5. Nakamura, A. and Tsukiji, S., "Ratiometric fluorescence imaging of nuclear pH in living cells using hoechst-tagged fluorescein.", Bioorg. Med. Chem. Lett., 27 (14), 3127~3130 (2017). [PMID:28558968]

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Video

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Product Information

[Date : November 06 2024 00:13]

Detail Product Name Product Code Supplier Size Price
NucleoSeeing, Live Nucleus Green
DatasheetThis may not be the latest data sheet.
FDV-0029 FNAFunakoshi Co.,Ltd. 0.1 mg $350

Description NucleoSeeing is a novel live cell imaging probe which emits green fluorescence by binding to DNA specifically. NucleoSeeing can be used for not only in animal cells or tissues, but also Guard cell of Arabidopsis thaliana with high S/N ratio. Moreover, NucleoSeeing can be used as a pH sensor in nucleus.
Storage -20°C CAS
Link

"NucleoSeeing" (Reversible Fluorescent Dye for Nucleus) in a minute!

[Date : November 06 2024 00:13]

NucleoSeeing, Live Nucleus Green


  • Product Code: FDV-0029
  • Supplier: FNA
  • Size: 0.1mg
  • Price: $350

Description NucleoSeeing is a novel live cell imaging probe which emits green fluorescence by binding to DNA specifically. NucleoSeeing can be used for not only in animal cells or tissues, but also Guard cell of Arabidopsis thaliana with high S/N ratio. Moreover, NucleoSeeing can be used as a pH sensor in nucleus.
Storage -20°C CAS
Link

"NucleoSeeing" (Reversible Fluorescent Dye for Nucleus) in a minute!




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