Biological Relevancy of Quantitative Analysis of Adherence Receptors on Polymorphonuclear.Neutrophil by Flow Cytometry and Optical Scanning Microscopy..

1999 ◽  
Vol 5 (S2) ◽  
pp. 1122-1123
Author(s):  
Dominique Dumas ◽  
Véronique Latger ◽  
Jean F. Stoltz
Keyword(s):  
Flow Cytometry ◽  
Molecular Conformation ◽  
Polymorphonuclear Neutrophils ◽  
Scanning Microscopy ◽  
Optical Scanning ◽  
Surface Molecules ◽  
Activation Level ◽  
Cellular Population ◽  
Necrosis Factor

Leukocytes adherence receptors are the basis of cells interactions1. Flow cytometry (FCM) makes it possible to assess a characterization of the activation level of a given cellular population by receptor quantifying2. That technique, however does not integrate at the same time, all others factors of leucocyte adhesive phenotype regulation, as spatial distribution and molecular conformation. Our study consisted in exploring the main adhesion receptors (CD62L, CDllb/CD18) on the surface of Polymorphonuclear Neutrophils (PMN) that were prepared under identical conditions and simultaneously analyzed by FCM and Conventional Optical Scanning Microscopy/Deconvolution3 (COSM). Methods: PMN cells from whole blood were obtained using Polymorphprep™. Isolated PMN (105 cells /ml) were incubated with Tumor Necrosis Factor (TNFα, 100 UI/ml) at 37°C for one hour. Quantification of cell surface molecules was assessed according to the quantitative indirect immunofluorescence3 method using QIFIKIT® (Dako, France).

scholarly journals KD determination from time-resolved experiments on live cells with LigandTracer and reconciliation with end-point flow cytometry measurements

2021 ◽  
Author(s):  
Diana Spiegelberg ◽  
Jonas Stenberg ◽  
Pascale Richalet ◽  
Marc Vanhove
Keyword(s):  
Flow Cytometry ◽  
Live Cells ◽  
Time Resolved ◽  
Surface Receptors ◽  
Full Characterization ◽  
End Point ◽  
Titration Curves ◽  
Kinetics Of

AbstractDesign of next-generation therapeutics comes with new challenges and emulates technology and methods to meet them. Characterizing the binding of either natural ligands or therapeutic proteins to cell-surface receptors, for which relevant recombinant versions may not exist, represents one of these challenges. Here we report the characterization of the interaction of five different antibody therapeutics (Trastuzumab, Rituximab, Panitumumab, Pertuzumab, and Cetuximab) with their cognate target receptors using LigandTracer. The method offers the advantage of being performed on live cells, alleviating the need for a recombinant source of the receptor. Furthermore, time-resolved measurements, in addition to allowing the determination of the affinity of the studied drug to its target, give access to the binding kinetics thereby providing a full characterization of the system. In this study, we also compared time-resolved LigandTracer data with end-point KD determination from flow cytometry experiments and hypothesize that discrepancies between these two approaches, when they exist, generally come from flow cytometry titration curves being acquired prior to full equilibration of the system. Our data, however, show that knowledge of the kinetics of the interaction allows to reconcile the data obtained by flow cytometry and LigandTracer and demonstrate the complementarity of these two methods.

scholarly journals Release of tumor necrosis factor by human polymorphonuclear leukocytes

Blood ◽  
1990 ◽  
Vol 76 (7) ◽  
pp. 1405-1409 ◽  
Author(s):  
JY Djeu ◽  
D Serbousek ◽  
DK Blanchard
Keyword(s):  
Tumor Necrosis Factor ◽  
Polymorphonuclear Leukocytes ◽  
Tumor Necrosis ◽  
Polymorphonuclear Neutrophils ◽  
Opportunistic Fungi ◽  
Produce Tumor Necrosis Factor ◽  
Human Polymorphonuclear Leukocytes ◽  
Block Protein Synthesis ◽  
Granular Lymphocytes ◽  
Necrosis Factor

Abstract Evidence is presented that human polymorphonuclear neutrophils (PMN) can be induced to produce tumor necrosis factor (TNF). Other investigators have previously reported that TNF has been induced from macrophages by bacteria and, more recently, from natural killer cells by certain tumor cells. Our laboratory has reported that the opportunistic fungi, Candida albicans, can induce TNF, not only from human monocytes, but also from Percoll-fractionated large granular lymphocytes. We now report that incubation of PMN with C albicans for 3 hours was sufficient for detection of TNF release, and peak induction was observed at 8 to 18 hours. This release was inhibitable by actinomycin D, an inhibitor of RNA synthesis, as well as by emetine and cycloheximide, which block protein synthesis. The TNF produced by PMN was neutralized by specific monoclonal antibodies against human TNF. These results represent an important finding that TNF production is a normal response of PMN to stimulation by fungi such as C albicans and suggest that the release of TNF may be related to autocrine activation of PMN effector function to control Candida growth.

Polarized light-scattering profile-advanced characterization of nonspherical particles with scanning flow cytometry

2011 ◽  
Vol 79A (7) ◽  
pp. 570-579 ◽  
Author(s):  
Dmitry I. Strokotov ◽  
Alexander E. Moskalensky ◽  
Vyacheslav M. Nekrasov ◽  
Valeri P. Maltsev
Keyword(s):  
Flow Cytometry ◽  
Light Scattering ◽  
Polarized Light ◽  
Advanced Characterization ◽  
Nonspherical Particles

T2050 Immunophenotypic Characterization of the Cellular Infiltrate During Murine Experimental Colitis Using Multiparameter Flow Cytometry

2010 ◽  
Vol 138 (5) ◽  
pp. S-621
Author(s):  
Kimberly A. Zins ◽  
Tamas Ordog ◽  
Michael R. Bardsley ◽  
Gianrico Farrugia ◽  
Joseph H. Szurszewski ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Experimental Colitis ◽  
Multiparameter Flow Cytometry ◽  
Cellular Infiltrate
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