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001-es BibID:	BIBFORM039658
035-os BibID:	(scopus)0036628631 (wos)000176656500002
Első szerző:	Sebestyén Zsolt
Cím:	Long wavelength fluorophores and cell-by-cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual-laser benchtop flow cytometer / Zsolt Sebestyén, Péter Nagy, Gábor Horváth, György Vámosi, Reno Debets, Jan W. Gratama, Denis R. Alexander, János Szöllősi
Dátum:	2002
ISSN:	0196-4763
Megjegyzések:	Flow cytometric fluorescence resonance energy transfer (FCET) is an efficient method to map associations between biomolecules because of its high sensitivity to changes in molecular distances in the range of 1-10 nm. However, the requirement for a dual-laser instrument and the need for a relatively high signal-to-noise system (i.e., high expression level of the molecules) pose limitations to a wide application of the method.METHODS:Antibodies conjugated to cyanines 3 and 5 (Cy3 and Cy5) were used to label membrane proteins on the cell surface. FCET measurements were made on a widely used benchtop dual-laser flow cytometer, the FACSCalibur, by using cell-by-cell analysis of energy transfer efficiency.ResultsTo increase the accuracy of FCET measurements, we applied a long wavelength donor-acceptor pair, Cy3 and Cy5, which beneficially affected the signal-to-noise ratio in comparison with the classic pair of fluorescein and rhodamine. A new algorithm for cell-by-cell correction of autofluorescence further improved the sensitivity of the technique; cell subpopulations with only slightly different FCET efficiencies could be identified. The new FCET technique was tested on various direct and indirect immunofluorescent labeling strategies. The highest FCET values could be measured when applying direct labeling on both (donor and acceptor) sides. Upon increasing the complexity of the labeling scheme by introducing secondary antibodies, we detected a decrease in the energy transfer efficiency.CONCLUSIONS:We developed a new FCET protocol by applying long wavelength excitation and detection of fluorescence and by refining autofluorescence correction. The increased accuracy of the new method makes cells with low receptor expression amenable to FCET investigation, and the new approach can be implemented easily on a commercially available dual-laser flow cytometer, such as a FACSCalibur.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Algorithms analysis Antibodies Biophysics Calibration Cell Line, Transformed Cells Comparative Study Dyes Energy Transfer Flow Cytometry Fluorescein Fluorescence Fluorescence Resonance Energy Transfer Fluorescent Dyes Humans Hungary instrumentation Lasers Membrane Proteins methods Proteins Reproducibility of Results Research Spectrometry, Fluorescence Subtraction Technique Support Tumor Cells, Cultured egyetemen (Magyarországon) készült közlemény
Megjelenés:	Cytometry. - 48 : 3 (2002), p. 124-135. -
További szerzők:	Nagy Péter (1971-) (biofizikus) Horváth Gábor (1974-) (biofizikus) Vámosi György (1967-) (biofizikus) Debets, Reno Gratama, Jan Willem Alexander, Denis R. Szöllősi János (1953-) (biofizikus)
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001-es BibID:	BIBFORM005192
035-os BibID:	(WOS)000257507300075
Első szerző:	Sebestyén Zsolt
Cím:	Human TCR that incorporate CD3 zeta induce highly preferred pairing between TCR alpha and beta chains following gene transfer / Zsolt Sebestyén, Erik Schooten, Tamara Sals, Irene Zaldivar, Esther San José, Balbino Alarcón, Sara Bobisse, Antonio Rosato, János Szöllősi, Jan Willem Gratama, Ralph A. Willemsen, Reno Debets
Dátum:	2008
ISSN:	0022-1767
Megjegyzések:	TCR gene therapy is adversely affected by newly formed TCR alpha beta heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCR alpha beta dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3 with specificities for three different Ags. Transfer of either TCR alpha:CD3 zeta or beta:CD3 zeta genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCR alpha beta:CD3 zeta does not compromise surface expression and functions of an endogenous TCR alpha beta. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCR alpha beta:CD3 zeta is the first strategy that results in highly preferred pairing between CD3 zeta-modified TCR alpha and beta chains as well as absence of TCR mispairing between TCR:CD3 zeta and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3 zeta chains is independent of endogenous CD3 gamma, 8 delta, and epsilon. Taken together, our data support the use of TCR alpha beta:CD3 zeta to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.
Tárgyszavak:	Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban folyóiratcikk Energy Transfer Flow Cytometry Fluorescence Fluorescence Resonance Energy Transfer Gene Therapy Human Support therapy
Megjelenés:	Journal of Immunology. - 180 : 11 (2008), p. 7736-7746. -
További szerzők:	Schooten, Erik Sals, Tamara Zaldivar, Irene San José, Esther Alarcón, Balbino Bobisse, Sara Rosato, Antonio Szöllősi János (1953-) (biofizikus) Gratama, Jan Willem Willemsen, Ralph A. Debets, Reno
Internet cím:	DOI Intézményi repozitóriumban (DEA) tárolt változat
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