Physical association between HLA class I and class II molecules detected on the cell surface by flow cytometric energy transfer

Szöllősi, János; Brodsky, FM; Balázs, Margit; Nagy, Péter; Trón, Lajos; Fulwyler, Mack J.; Damjanovich, Sándor

doi:10.1016/0198-8859(88)90270-4

Cited by 19 publications

(39 citation statements)

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“…Antibodies used as donoracceptor pairs did not mask or unmask each other since the binding of any of these antibodies was not affected by prior incubation of the cells with another unlabeled antibody. Aliquots of purified monoclonal antibodies and Fab fragments were labeled with fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC, Molecular Probes, Eugene, OR) as described (41). (Use of capital F or R in front of the name of an antibody or Fab fragment refers to fluoresceinated and rhodaminated antibodies, respectively.)…”

Section: Antibodiesmentioning

confidence: 99%

EGF-induced redistribution of erbB2 on breast tumor cells: Flow and image cytometric energy transfer measurements

et al. 1998

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Section: Antibodiesmentioning

confidence: 99%

EGF-induced redistribution of erbB2 on breast tumor cells: Flow and image cytometric energy transfer measurements

et al. 1998

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“…Fluorochrome conjugated monoclonal antibodies against the heavy chain and 02-microglobulin components of class I MHC molecules revealed a close proximity of these two antibody-binding sites using Forster-type resonance energy transfer technique (58).…”

Section: Lretq Between Heavy and Light Chains Of Class I Mhc M O L E mentioning

confidence: 99%

Luminescence quenching by long range electron transfer: A probe of protein clustering and conformation at the cell surface

et al. 1995

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Quenching of luminescence from fluorescent and phosphorescent probes by nitroxide spin labels with a long range electron transfer (LRET) mechanism (44,45) has been tested as a tool to monitor associatiodclustering and conformational changes of cell surface proteins. The membrane proteins were labeled with monoclonal antibodies or Fab fragments conjugated with luminescent probes or water-soluble nitroxide spin labels. The method was tested as a probe of 3 different aspects of protein-protein association involving class I MHC molecules: (1) interaction between the heavy and light chains of the MHC molecules, (2) clustering, selfassociation of MHC molecules, (3) proximity of MHC molecules to transferrin receptors of tibroblasts or surface immunoglobulin molecules of B lymphoblasts. The extent of quenching upon increasing the fiactlonal density of the quencher was sensitive for protein association in accordance with earlier immunoprecipitation and flow cytometric Forster-type energy transfer (FCET) data obtained on the same cells. These data suggest that the LRET quenching can be used as intra-or intermolecular ruler in a 0.5-2.5 nm distance range. This approach is simpler (measurements only on donor side) and h t e r than many other experimental techniques i n screening physical association or conformational changes of membrane proteins by means of spectrofluorimetry, flow cytometry, or microscope based imaging. o 1995 Wfley-Liss, I~C .Key terms: Fluorescence quenching, protein association, clusters, electron transfer, flow cytometry Association of membrane proteins may result in formation of local assemblies, clusters of proteins on the cell surface, or in organelle membranes (9,10,13,15,16, 26-28). These assemblies may be relatively short-lived, induced by external triggering molecules ( 14,15,55), or long-lived, mediated by cytoskeleton ( 4 ) or by membrane cholesterol ( 5 2 ) . Formation and persistence of some assemblies may be genetically determined ( 9 5 5 4 ) . There are several examples of functional significance of protein clustering, particularly in immunology (5-Given the functional significance of protein clusters it is important to probe their formation and dissolution in living cells. Due to the resolution limits of conventional light microscopy, the majority of current techniques for detecting protein clusters on living cells are spectroscopic and based on variations of Forster-type (dipoledipole) resonance energy transfer ( 2 1,3 1-33,43,46,56, 57,60,63). The efficiency of Forster-type energy transfer, a parameter dependent upon proximity of donor-and acceptor-bearing molecules, is determined from either steady-state or time-resolved intensity measurements or showed that quenching of various luminescent probes by water soluble nitroxide quenchers is dominated by a long range electron transfer (LRET) mechanism. This finding, in accordance with other recent LRET observations ( 1,29,36,4 1,45,64), suggests that LRET between electron donors and acceptors carried by Fab fragments of monoclonal antibodi...

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“…JY and HUT-102B2 cell lines, originally derived from a human adult T and a B lymphoma, respectively, were cultured similarly in RPMI-1640 medium supplemented with 10% bovine calf serum and antibiotics (61,62). The Kit K6 cell line (obtained from Takashi Uchiyama, Kyoto, Japan) that was originally derived from a human adult T cell lymphoma was cultured as the cell lines above, but it was also supplemented with 20 units/ml of recombinant interleukin (IL)-2, administered on a daily basis.…”

Section: Materials and Methods Cellsmentioning

confidence: 99%

“…The fluorescence was gated on the forward angle light scatter signal to avoid artifacts from debris. The energy transfer frequency distribution curves were calculated from the equations described previously (17,54,55,58,62,68,69). These equations corrected the energy transfer data for the direct excitability of the acceptor at the donor's excitation wavelength, and the spill-over of the donor's fluorescence at the detection site of the energy transfer by the aid of computer program named FloWin (beta 2), which was calculated from data measured for each cell.…”

Section: Flow Cytometric Energy Transfer Measurementsmentioning

confidence: 99%

“…The labeling of mAbs was carried out as before (37,61,62). Briefly, the covalent binding of isothiocyanate derivatives of fluorescein, or tetramethyl rhodamine (Molecular Probes, Leiden, The Netherlands) and the sulfoindocyanine succinimidyl bifunctional ester derivative of Cy3 (Amersham, Braunschweig, Germany) to lysyl-⑀-amino groups of the antibodies was carried out in bicarbonate buffer at pH 9.…”

Section: Conjugation Of Mabs and Fab Fragments With Fluorescent Dyesmentioning

confidence: 99%

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Supramolecular receptor structures in the plasma membrane of lymphocytes revealed by flow cytometric energy transfer, scanning force- and transmission electron-microscopic analyses

et al. 1998

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Physical association between HLA class I and class II molecules detected on the cell surface by flow cytometric energy transfer

Cited by 19 publications

References 0 publications

EGF-induced redistribution of erbB2 on breast tumor cells: Flow and image cytometric energy transfer measurements

EGF-induced redistribution of erbB2 on breast tumor cells: Flow and image cytometric energy transfer measurements

Luminescence quenching by long range electron transfer: A probe of protein clustering and conformation at the cell surface

Supramolecular receptor structures in the plasma membrane of lymphocytes revealed by flow cytometric energy transfer, scanning force- and transmission electron-microscopic analyses

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