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Imaging of neurosphere oxygenation with phosphorescent probes

Paper ID Volume ID Publish Year Pages File Format Full-Text
6441 491 2013 11 PDF Available
Title
Imaging of neurosphere oxygenation with phosphorescent probes
Abstract

Multicellular spheroids are useful models of mammalian tissue for studies of cell proliferation, differentiation, replacement therapies and drug action. Having a size of 100–500 μm they mimic in vivo micro-environment and characteristic gradients of O2, pH and nutrients. We describe the use of cell-penetrating O2 probes based on phosphorescent Pt-porphyrins to perform high-resolution 2D and 3D mapping of O2 in spheroid structures by live cell fluorescence imaging technique. Optimised procedures for preparation of neurospheres from cortical neural cells isolated from embryonic rat brain, their staining with the phosphorescent O2 probes NanO2 and MM2 and subsequent analysis of oxygenation on different live cell imaging platforms, including widefield and confocal phosphorescence lifetime imaging microscopy (PLIM), conventional confocal and two-photon ratiometric intensity based O2 detection are presented. This is followed by a series of physiological experiments in which oxygenation patterns of the neurospheres are correlated with culturing conditions (atmospheric hypoxia and hyperoxia, size, growth factors), distribution of stem cells, mature neurons and astrocytes, HIF-2α stabilisation and responses to metabolic stimulation. The O2 imaging method allows multiplexing with many conventional fluorescent probes to perform multi-parametric imaging analysis of cells in 3D microenvironment. It can be applied to other types of spheroids and 3D tissue models.

Keywords
3D tissue model; Multi-cellular spheroids; Stem cell; Phosphorescent O2 sensitive probe; Oxygen imaging; PLIM3D, three-dimensional; CTX, cholera toxin; DIV, days in vitro; FLIM, fluorescence lifetime imaging microscopy; HBSS, Hank's balanced salt solution
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomaterials - Volume 34, Issue 37, December 2013, Pages 9307–9317
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us