Flow cytometry: A novel approach for the quantitative analysis of receptor-ligand interactions on surfaces of living cells

Bohn, B.

doi:10.1016/0303-7207(80)90090-8

Cited by 40 publications

(8 citation statements)

References 13 publications

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“…Many types of additional analysis are possible given the appropriate fluorescent probes. For example, flow cytometric methods are well suited to receptor binding studies (for review, see Bohn, 1980) and have been used to study the cellular binding of a number of fluorescently labeled ligands, including a chemotactic peptide, estrogens, and luteinizing hormone-releasing hormone (Sklar and Finney, 1982;Kute et al, 1983;MacInnes et al, 1983;Oxenhandler et al, 1984;Benz et al, 1985). Since 2 or more fluorescence signals can be analyzed simultaneously, it should be possible to characterize and sort nerve terminals based on the coincidence of several cell surface and/or intracellular molecules.…”

Section: Discussionmentioning

confidence: 99%

Flow cytometric analysis of rat striatal nerve terminals

Wolf

Kapatos

1989

J. Neurosci.

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Methods were developed for the analysis and isolation of striatal nerve terminals (synaptosomes) using fluorescence-activated cell sorting (FACS). Comparison of the light-scattering properties of synaptosomal and mitochondrial fractions indicated that particles in the synaptosomal fraction were generally larger and more sensitive to hypotonic lysis, consistent with results obtained by other methods of analysis. FACS analysis using indirect immunofluorescence techniques indicated that approximately 84% of the synaptosomal fraction was labeled by monoclonal antibody (mAb) A2B5 and thus appeared to be of neuronal origin. After permeabilization, between 5 and 10% of the particles were labeled by a mAb to glial fibrillary acidic protein, suggesting that they were derived from astrocytes. A fluorescent voltage-sensitive dye (VSD) was used to distinguish intact synaptosomes from free mitochondria (only the former maintain a membrane potential under the present experimental conditions). Approximately 83% of the synaptosomal fraction exhibited increased fluorescence after incubation with the VSD; furthermore, the fluorescence signal decreased in response to depolarizing agents (elevated potassium and veratridine). A portion of the mitochondrial fraction responded similarly, consistent with the presence of contaminating synaptosomes. Analysis of synaptosomal labeling by 11 fluorescein-conjugated plant lectins indicated that striatal nerve terminals differ significantly in their cell surface glycoconjugates. Subpopulations of synaptosomes defined on the basis of lectin binding were collected by FACS onto filters and probed with a mAb to tyrosine hydroxylase (TH) using Western blot techniques. While subpopulations exhibited different amounts of TH immunoreactivity, none of the lectins appeared to recognize TH-positive (i.e., dopaminergic) synaptosomes exclusively. These findings demonstrate that synaptosomes can be characterized and isolated for further study based on FACS analysis of properties such as size, membrane potential, and the presence of intracellular or cell surface molecules.

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Section: Discussionmentioning

confidence: 99%

Flow cytometric analysis of rat striatal nerve terminals

Wolf

Kapatos

1989

J. Neurosci.

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“…The effect of conjugation of LDL with Dil or RITC on their ability to compete with native LDL was studied as proposed by Bohn. 49 Monocytes were incubated in a constant volume of medium and a constant mass of total LDL (10 i±g) but with varying relative amounts of fluorescence-labeled LDL (0% to 100% affinity for the LDL receptor (Fig 2D). Specific binding of Dil LDL and I25 I-LDL was also compared and calculated as the difference of total and nonspecific binding.…”

Section: Binding Characteristics Of Dil Ldl On Human Monocytesmentioning

confidence: 99%

Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia.

Schmitz

Brüning

Kovács

et al. 1993

Arterioscler Thromb

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A flow-cytometric method with fluorescence-labeled monoclonal antibodies (MABs) against the low density lipoprotein (LDL) receptor (C7A MAB) or 3,3'-dioctadecylindocarbocyanin-iodide (Dil) LDL has been developed that allows the quantification of LDL receptors on leukocytes and the identification of patients with familial hypercholesterolemia (FH) within 48 hours. Leukocytes were isolated from 10 mL anticoagulated blood by density gradient centrifugation. To induce maximal expression of LDL receptors, mononuclear cells were preincubated with either phytoheraagglutinine (PHA) or iipoprotein-deflcient serum (LPDS). LPDS-treated raonocytes provided a more homogeneous cell population with regard to LDL receptor activity than did the PHA-treated lymphocytes; they also provided a greater discrimination between the fluorescence of the receptor probes and cellular autofluorescence. The C7A MAB was able to compete for Dil LDL binding by about 40%. In competition with unlabeled LDL, Dil LDL revealed linear binding, indicating an affinity similar to native LDL. The binding characteristics of Dil LDL were also similar to IZ5 I-LDL binding. LDL isolated from familial defective apolipoprotein B-100 was not able to compete for Dil LDL binding on monocytes, whereas native LDL reduced it by about 80%. In monocytes from FH heterozygous patients, the cellular mean fluorescence using either C7A MAB or Dil LDL at 4°C was 30% to 70%; in FH homozygotes, cellular mean fluorescence was less than 20% of that in monocytes from normal individuals. In patients with familial defective apolipoprotein B-100 antibody binding was normal, but one patient's own LDL failed to compete with normal Dil LDL for 4°C binding on U937 test monocytes. Patient monocytes having internalization defects showed normal 4°C Dil LDL binding, but at 20°C cell-associated fluorescence was reduced by about 40%. In our study 384 hypercholesterolemic patients (preselected according to serum cholesterol levels, clinical symptoms, and family history) were analyzed for LDL receptor expression using the C7A MAB-based assay. In 71.8% of the patients with cholesterol levels higher than 300 mg/dL, an LDL receptor deficiency was observed. Apolipoprotein E isofonns and lipoprotein [a] were found to be independent from the LDL receptor status. In some patients with high cholesterol levels but normal LDL receptor expression with the C7A MAB assay, LDL receptor defects could be diagnosed when either reduced binding or internalization of Dil LDL or familial defective apolipoprotein B-100 was detected. We conclude that fluorescence flow cytometry provides an appropriate, easily performed assay system for the differential diagnosis of LDL receptor defects, including LDL receptor deficiencies and internalization defects, and also allows the discovery of ligand defects.

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“…Flow cytometry (FCM) is a method used to detect the presence and amount of cellular constituents in large numbers of single cells allowing for the enumeration and functional characterization of different cell types in complex cellular mixtures (9, 22, 23, 30, 46, 52). FCM can be used to measure the expression of membrane proteins, intracellular and secreted cell constituents, nucleic acids, phospholipids and post-translationally modified forms of proteins allowing FCM to be used to monitor cellular processes like DNA replication, programmed cell death and signal transduction activation (14, 18, 21, 24, 25, 27, 35).…”

Section: Introductionmentioning

confidence: 99%

Elucidation of seventeen human peripheral blood B‐cell subsets and quantification of the tetanus response using a density‐based method for the automated identification of cell populations in multidimensional flow cytometry data

Wei

Lee

et al. 2010

Cytometry Part B Clinical

187

214

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Background Advances in multi-parameter flow cytometry (FCM) now allow for the independent detection of larger numbers of fluorochromes on individual cells, generating data with increasingly higher dimensionality. The increased complexity of these data has made it difficult to identify cell populations from high-dimensional FCM data using traditional manual gating strategies based on single-color or two-color displays. Methods To address this challenge, we developed a novel program, FLOCK (FLOw Clustering without K), that uses a density-based clustering approach to algorithmically identify biologically relevant cell populations from multiple samples in an unbiased fashion, thereby eliminating operator-dependent variability. Results FLOCK was used to objectively identify seventeen distinct B cell subsets in a human peripheral blood sample and to identify and quantify novel plasmablast subsets responding transiently to tetanus and other vaccinations in peripheral blood. FLOCK has been implemented in the publically available Immunology Database and Analysis Portal – ImmPort (http://www.immport.org) for open use by the immunology research community. Conclusions FLOCK is able to identify cell subsets in experiments that use multi-parameter flow cytometry through an objective, automated computational approach. The use of algorithms like FLOCK for FCM data analysis obviates the need for subjective and labor intensive manual gating to identify and quantify cell subsets. Novel populations identified by these computational approaches can serve as hypotheses for further experimental study.

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Flow cytometry: A novel approach for the quantitative analysis of receptor-ligand interactions on surfaces of living cells

Cited by 40 publications

References 13 publications

Flow cytometric analysis of rat striatal nerve terminals

Flow cytometric analysis of rat striatal nerve terminals

Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia.

Elucidation of seventeen human peripheral blood B‐cell subsets and quantification of the tetanus response using a density‐based method for the automated identification of cell populations in multidimensional flow cytometry data

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