Analysis of fluorescence lifetime and quenching of FITC-conjugated antibodies on cells by phase-sensitive flow cytometry

Deka, Chiranjit; Lehnert, Bruce E.; Lehnert, Nancy M.; Jones, Greg; Sklar, Larry A.; Steinkamp, John A.

doi:10.1002/(sici)1097-0320(19961101)25:3<271::aid-cyto8>3.0.co;2-i

Cited by 86 publications

(74 citation statements)

References 17 publications

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“…Resonance energy transfer between fluorescein molecules should also be observable by fluorescence lifetime measurements; i.e., a decrease in lifetimes of excited states should be observed at high labeling densities [18,21,43]. We measured and compared fluorescence lifetimes for coreshell NPs and nanoshells using TCSPC.…”

Section: Resultsmentioning

confidence: 99%

Reduction of Self-Quenching in Fluorescent Silica-Coated Silver Nanoparticles

et al. 2008

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This paper reports the development of spherical Ag@SiO 2 nanocomposites in which fluorescein isothiocyanate molecules have been incorporated using a silane coupling agent and a straightforward microemulsion-based synthesis procedure. The photophysical characteristics of core-shell and coreless nanostructures with similar silica shell thickness and fluorophore densities are measured and compared, and show unequivocally that the presence of the silver core decreases the fluorophore lifetime by a factor as high as 4 and that the steady-state fluorescence intensity is increased by a factor as high as 3. The relationship between the enhancement in fluorescence yield and the influence of the silver core on resonance energy transfer processes was examined by fluorescence lifetime and anisotropy measurements. These Ag@SiO 2 core-shell nanoparticles provide higher detectability and lower self-quenching, whereas the faster recycling time offers more robustness toward photobleaching.

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Section: Resultsmentioning

confidence: 99%

Reduction of Self-Quenching in Fluorescent Silica-Coated Silver Nanoparticles

et al. 2008

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“…We used phycoerythrin (PE)-and allophycocyanin (APC)-conjugated mAbs for quantification because they do not self-quench at high density (11,12). Tricolor (Tri) and FITC were the two other fluorochromes used in our four-color FACS analysis.…”

Section: Methodsmentioning

confidence: 99%

Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages

Lee

Sharron

Montaner

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

532

505

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CCR5 and CXCR4 are the major HIV-1 coreceptors for R5 and X4 HIV-1 strains, respectively, and a threshold number of CD4 and chemokine receptor molecules is required to support virus infection. Therefore, we used a quantitative fluorescence-activated cell sorting assay to determine the number of CD4, CCR5, and CXCR4 antibody-binding sites (ABS) on various T cell lines, T cell subsets, peripheral blood dendritic cells (PBDC), and monocyte-derived macrophages by using four-color fluorescence-activated cell sorting analysis on fresh whole blood. Receptor levels varied dramatically among the various subsets examined and typically varied from 2-to 5-fold between individuals. CCR5 was expressed at much higher levels in CD4؉͞CD45RO؉͞CD62L-true memory cells compared with CD4؉͞CD45RO؉͞CD62L؉ cells. Fresh PBDC had the highest number of CCR5 ABS among the leukocyte subsets examined but had few CXCR4 ABS, affording a strategy for sort-purifying PBDC. In vitro maturation of PBDC resulted in median 3-and 41-fold increases in CCR5 and CXCR4 ABS, respectively. We found that macrophage colony-stimulating factor caused the greatest up-regulation of both CCR5 and CXCR4 on macrophage maturation (from Ϸ5,000 to Ϸ50,000 ABS) whereas granulocytemacrophage colony-stimulating factor caused a marked decrease of CXCR4 (from Ϸ5,000 ABS to <500) while up-regulating CCR5 expression (from Ϸ5,000 to Ϸ20,000 ABS). Absolute ABS for CD4 and the major HIV-1 coreceptors serve as a more quantitative measure of cell surface expression, and we propose that this be used for future studies looking at the modulation of CD4 or chemokine receptor expression by cytokines, HIV-1 infection, or receptor polymorphisms.HIV-1 entry into cells requires sequential interactions between envelope (Env), CD4, and a coreceptor (1-3). Epidemiological and experimental evidence indicates that CD4 and coreceptor levels affect the efficiency of viral entry and that this may have consequences for the pathogenesis of HIV disease. Individuals homozygous for the ⌬32-ccr5 allele have no surface expression of CCR5 and are highly protected against HIV-1 infection, whereas ⌬32-ccr5 heterozygotes have lower CCR5 expression levels and progress to AIDS more slowly than individuals without this allele (reviewed in ref. 4). Individuals homozygous for a mutation in the SDF-1 gene also progress more slowly to clinical AIDS (5), perhaps because of increased expression of SDF-1 and modulation of CXCR4 expression. Indeed, in vitro studies have shown that CD4, CCR5, and CXCR4 expression levels impact the efficiency of viral entry (6-8).Chemokine receptor expression in both peripheral blood lymphocytes and monocyte-derived macrophages (MDM) is sensitive to cytokine-mediated modulation (reviewed in ref. 9). Because the presence of CD4 and either CCR5 and͞or CXCR4 on specific leukocytes and MDMs designates these cells as potentially susceptible targets for viral infection, it is important to determine quantitatively the amount of CD4 and the major coreceptors present on various leukocyte a...

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“…The self quenching of fluorescein derivatives (FlAsH is a fluorescein derivative) was already used successfully to study protein-protein and protein-lipid interactions (18,38,61).…”

Section: Cells Coexpressing Egfp-vpr and Pr55mentioning

confidence: 99%

HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

Fritz

Dujardin

Godet

et al. 2010

J Virol

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During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55Gag . Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G 2 /M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55Gag and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55Gag -TC. This TC motif is tethered to the C terminus of Pr55 Gag and does not interfere with Pr55Gag trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55 Gag -Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55Gag -TC mutants confirm that the 41 LXXLF domain of Gag-p6 is essential for Pr55 Gag -Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55Gag recognition and its accumulation at the plasma membrane. On the other hand, Pr55Gag -Vpr complexes are still formed when Pr55 Gag carries mutations impairing its multimerization. These findings suggest that Pr55Gag -Vpr recognition and complex formation occur early during Pr55Gag assembly.

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Analysis of fluorescence lifetime and quenching of FITC-conjugated antibodies on cells by phase-sensitive flow cytometry

Cited by 86 publications

References 17 publications

Reduction of Self-Quenching in Fluorescent Silica-Coated Silver Nanoparticles

Reduction of Self-Quenching in Fluorescent Silica-Coated Silver Nanoparticles

Quantification of CD4, CCR5, and CXCR4 levels on lymphocyte subsets, dendritic cells, and differentially conditioned monocyte-derived macrophages

HIV-1 Vpr Oligomerization but Not That of Gag Directs the Interaction between Vpr and Gag

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