Characterization of interaction between DNA and 4',6-diamidino-2-phenylindole by optical spectroscopy

Kubista, Mikael; Åkerman, Björn; Nordén, Bengt

doi:10.1021/bi00388a057

Cited by 224 publications

(160 citation statements)

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“…Indeed, we experienced inconsistency attempting to track EGFP-and LacZ-labeled MSCs. Our results DAPI is known to interact with AT clusters in the minor groove of double-stranded DNA (27), and this show that the DAPI label can be stably retained in the implanted MSCs for at least 1 month after implantation, property of DAPI may be expected to have some selective effects on gene expression. Indeed, we show that and these labeled MSCs can be detected histologically with little ambiguity.…”

Section: Discussionsupporting

confidence: 54%

Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

et al. 2008

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Therapeutic implantation of mesenchymal stem cells (MSCs) is entering the realm of clinical trials for several human diseases, and yet much remains uncertain regarding their dynamic distribution and cell fate after in vivo application. Discrepancies in the literature can be attributed in part to the use of different cell labeling/tracking methods and cell administration protocols. To identify a stem cell detection method suitable for myocardial implantation in a large animal model, we experimented on three different MSC labeling methods: adenovirus-mediated expression of enhanced green fluorescence protein (EGFP) and β-galactosidase (LacZ), and nuclear staining with DAPI. Intramuscular and intracoronary administrations of labeled porcine MSCs identified the nuclear affinity dye to be a reliable stem cell tracking marker. Stem cell identification is facilitated by an optimized live cell labeling condition generating bright blue fluorescence sharply confined to the nucleus. DAPI-labeled MSCs retained full viability, ceased proliferation, and exhibited an increased differentiation potential. The labeled MSCs remained fully active in expressing key growth factor and cytokine genes, and notably exhibited enhanced expression of the chemokine receptor CXCR4 and its ligand SDF1, indicating their competency in response to tissue injury. Histological analysis revealed that approximately half a million MSCs or ϳ2% of the administered MSCs remained localized in the normal pig heart 2 weeks after coronary infusion. That the vast majority of these identified MSCs were interstitial indicated the ability of MSCs to migrate across the coronary endothelium. No evidence was obtained indicating MSC differentiation to cardiomyocyte.Key words: Mesenchymal stem cells; Heart; Tracking; Implantation INTRODUCTIONfusion, and intercellular communication through GAP junctions or nanotubes have been proposed to account for the seeming pluripotency of adult stem cells in variStem cell therapy has in recent years received much attention in the field of regenerative medicine. Theraous implantation studies (26,32,50,53,62). Technically, these discrepancies may be caused in part by the use of peutic implantation of mesenchymal stem cells (MSCs), in particular, has been attempted in several clinical trials different cell labeling/tracking methods, cell administration protocols, timing of cell transplantation after tissue for hematological disorders, cardiovascular diseases, osteogenesis imperfecta, neurological pathologies, and even injury, and possibly misinterpretation of microscopy results (4). cancer therapy (9,14,23). The ease of isolation, capacity for large-scale expansion, and alleged immunoprivileged Many cell labeling techniques have been used for tracking implanted stem cells in vivo. Fluorescent quanstatus of MSCs (3,21,29) are likely to generate more clinical applications in the future. Central to these efforts tum dots and iridium nanoparticles are randomly internalized by cells during culturing (13,41). Membrane is a thorough unders...

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Section: Discussionsupporting

confidence: 54%

Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

et al. 2008

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“…After completion of phenol degradation, MRCs in the suspension were stained by 4 0 ,6-diamidino-5-phenyl-indole (DAPI) (Kubista et al, 1987) and counted under a Zeiss Axioplan 2 epifluorescence microscope. MRCs in the suspension were centrifuged at 3000 r.p.m.…”

Section: Mnp-mediated Cell Isolation and Countingmentioning

confidence: 99%

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community

et al. 2014

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Although uncultured microorganisms have important roles in ecosystems, their ecophysiology in situ remains elusive owing to the difficulty of obtaining live cells from their natural habitats. In this study, we employed a novel magnetic nanoparticle-mediated isolation (MMI) method to recover metabolically active cells of a group of previously uncultured phenol degraders, Burkholderiales spp., from coking plant wastewater biosludge; five other culturable phenol degraders-Rhodococcus sp., Chryseobacterium sp. and three different Pseudomonas spp.-were also isolated from the same biosludge using traditional methods. The kinetics of phenol degradation by MMI-recovered cells (MRCs) was similar to that of the original sludge. Stable isotope probing (SIP) and pyrosequencing of the 16S rRNA from the 'heavy' DNA ( 13 C-DNA) fractions indicated that Burkholderiales spp. were the key phenol degraders in situ in the biosludge, consistent with the results of MRCs. Single-cell Raman micro-spectroscopy was applied to probe individual bacteria in the MRCs obtained from the SIP experiment and showed that 79% of them were fully 13 C-labelled. Biolog assays on the MRCs revealed the impact of various carbon and nitrogen substrates on the efficiency of phenol degradation in the wastewater treatment plant biosludge. Specifically, hydroxylamine, a metabolite of ammonia oxidisation, but not nitrite, nitrate or ammonia, inhibited phenol degradation in the biosludge. Our results provided a novel insight into the occasional abrupt failure events that occur in the wastewater treatment plant. This study demonstrated that MMI is a powerful tool to recover live and functional cells in situ from a complex microbial community to enable further characterisation of their physiology.

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“…r 02 ) r 01 3 cos 2 ϑ 12 -1 2 (6) distance of 8 Å from the helical axis (Kubista et al, 1987). Under these assumptions, only one angle, the angle φ, is sufficient to determine the orientation of the DAPI molecule, i.e., the angle with the cylinder axis ( Figure 1).…”

Section: Depolarization Due To Energy Transfermentioning

confidence: 99%

“…In addition, several DNA dye complexes have been crystallized, and the X-ray structure has been determined (Jain & Sobell, 1984;Pjiura et al, 1987;Ginell et al, 1988;Larsen et al, 1989;Spink et al, 1994). A common probe for DNA studies is DAPI, a minor groove binding probe, and progress has been made toward a better understanding of the modalities of binding for this probe (Kapuscinski & Szer, 1979;Manzini et al, 1983Manzini et al, , 1985bWilson et al, 1985aWilson et al, , 1989Szabo et al, 1986;Kubista et al, 1987;Neidle et al, 1987;Strekowski et al, 1989;Hard et al, 1990;Loontiens et al, 1991;Jansen et al, 1993). In particular, we have reached a partial consensus about the photophysical and photochemical deactivation processes for the DNA/DAPI complex (Cavatorta et al, 1985;Barcellona & Gratton, 1991;Tanious et al, 1992).…”

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confidence: 98%

Torsional Dynamics and Orientation of DNA−DAPI Complexes

Barcellona

Gratton

1996

Biochemistry

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The flexibility of calf thymus DNA and several polynucleotides was measured using the anisotropy decay of DAPI bound to DNA, a minor groove probe. DNA torsional dynamics were analyzed using the Schurr model [Allison, S. A., & Schurr, J. M. (1979) Chem. Phys. 41, 35-44] in the infinite polymer length approximation. Time-resolved fluorescence depolarization was measured using a frequencydoubled mode-locked dye laser and frequency-domain acquisition methods. At very high P/D ratios, the anisotropy decay is dominated by DNA torsional dynamics. The recovered values of the torsional elastic constant were in good agreement with literature values obtained using other DNA probes. The exact knowledge of the angle between the probe emission dipole transition moment and the helix axis is critical for the determination of the polymer elastic constant. At low P/D ratios, energy transfer between dye molecules strongly contributes to the anisotropy decay. We have developed a statistical model that describes the anisotropy decay, when the correct geometrical factors are included. At low P/D ratios the anisotropy decay is dominated by fluorescence homotransfer. In this regime, it is possible to determine the orientation of the dye molecule with respect to the polymer with accuracy. The values obtained for the distance and orientation of the DAPI molecules in solution using the fluorescence measurements are in excellent agreement with those from the crystal structure of the oligonucleotides-DAPI complex by Dickerson's group [Larsen T.

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Characterization of interaction between DNA and 4',6-diamidino-2-phenylindole by optical spectroscopy

Cited by 224 publications

References 39 publications

Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

Magnetic nanoparticle-mediated isolation of functional bacteria in a complex microbial community

Torsional Dynamics and Orientation of DNA−DAPI Complexes

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