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Method Article
David Hevia
Nutraceuticals and cancer
Corresponding Author
Juan C Mayo
Nutraceuticals and cancer
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO), a molecule taken up by living adherent cells. Dual binding of AO to either DNA or RNA allows complete staining. When combined with confocal microscopy, 3D software can be used for in vivo living cell reconstruction. Beside the purpose that we intend here, a fast and easy system for cell volume determination, the protocol is an easy approach to study changes in morphology during cellular processes such as cell differentiation. Novel therapeutic approaches would require some knowledge about how these drugs enter into cells/tissues. For this purpose fast and accurate in vivo cell volume determinations such as the method reported here, in combination with analytical methods, would allow estimating intracellular concentrations of compounds and might be further employed for finding out whether any new drug can reach the effective concentration inside its cellular target. Furthermore this protocol with minimal adjustments will permit the determination of morphometric parameters in vivo in different types of adherent cells.
Keywords
3D, cell volume, laser scanning confocal microscopy, intracellular concentration
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The authors declare no competing financial interests
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.mov
Video 2 LNCaP cell <iframe width="420" height="315" src="http://www.youtube.com/embed/METM364pA18" frameborder="0" allowfullscreen></iframe>
- supplement0.mov
Video 3 PC3 cell <iframe width="420" height="315" src="http://www.youtube.com/embed/SVcUeDrbb8s" frameborder="0" allowfullscreen></iframe>
- supplement0.mov
Video 5 PNT1A cell <iframe width="420" height="315" src="http://www.youtube.com/embed/smQLoQQQ_Rs" frameborder="0" allowfullscreen></iframe>
- supplement0.mov
Video 4 CHO cell <iframe width="420" height="315" src="http://www.youtube.com/embed/myD_HyaVnJk" frameborder="0" allowfullscreen></iframe>
- supplement0.mov
Video 6 PC3 cells Video 6 shows a 360° rotation of PC3 cell around other cells <iframe width="420" height="315" src="http://www.youtube.com/embed/TBacWtA5E_Q" frameborder="0" allowfullscreen></iframe>
- supplement0.wmv
Video 1 Image data preparation Steps for processing imaging (steps 17-26) <iframe width="420" height="315" src="http://www.youtube.com/embed/uyzFPKJBa7s" frameborder="0" allowfullscreen></iframe>
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Version History
CURRENT STATUS: Posted
Version 1
Posted 08 Dec, 2011
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Subject Areas
Biological techniques
Method Article
David Hevia
Nutraceuticals and cancer
Corresponding Author
Juan C Mayo
Nutraceuticals and cancer
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 08 Dec, 2011
No community comments so far
Metrics
Comments: 0
HTML Views: ...
Subject Areas
Biological techniques
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
The protocol reported here describes a simple, easy, fast and reproducible method aimed to know the geometric parameters of living cells based on confocal laser scanning microscopy combined with 3D reconstruction software. Briefly, the method is based on intrinsic fluorescence properties of acridine orange (AO), a molecule taken up by living adherent cells. Dual binding of AO to either DNA or RNA allows complete staining. When combined with confocal microscopy, 3D software can be used for in vivo living cell reconstruction. Beside the purpose that we intend here, a fast and easy system for cell volume determination, the protocol is an easy approach to study changes in morphology during cellular processes such as cell differentiation. Novel therapeutic approaches would require some knowledge about how these drugs enter into cells/tissues. For this purpose fast and accurate in vivo cell volume determinations such as the method reported here, in combination with analytical methods, would allow estimating intracellular concentrations of compounds and might be further employed for finding out whether any new drug can reach the effective concentration inside its cellular target. Furthermore this protocol with minimal adjustments will permit the determination of morphometric parameters in vivo in different types of adherent cells.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
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