Use of anti-CD3/CD28 mAb coupled magnetic beads permitting subsequent phenotypic analysis of activated human T cells by indirect immunofluorescence.
Functional analysis of T lymphocytes requires in vitro stimulation of these cells under experimental conditions that mimic as closely as possible physiological in vivo stimulation and that involve antigen/T cell receptor (TCR)-mediated activation. Because of the low frequency of antigen-specific T cells in human clinical samples, stimulation with a combination of anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) is a preferred method. Interaction of these mAbs with their ligand results in modulation of the mAb-ligand complex from the cell surface. However, as a result of incomplete modulation, CD3/CD28 mAb complexes often remain at the cell surface, thereby precluding subsequent indirect immunofluorescence and flow cytometry analysis using mouse immunoglobulin (Ig)-specific antibodies. This is of importance in situations in which no specific fluorochrome-conjugated mAbs are available, such as in screening procedures of Ig-containing hybridoma culture supernatants. We propose here the use of CD3/CD28 mAbs, linked to magnetic beads allowing standardization of the activation conditions, optimal activation of T cells and complete modulation of antigen-antibody complexes from the cell surface.
Pène
Jérôme
J
INSERM U454, CHU Arnaud de Villeneuve, 371 Avenue Doyen Gaston Giraud, 34295 Cedex 5, Montpellier, France.
Rahmoun
Massilva
M
Temmerman
Stéphane
S
Yssel
Hans
H
D036541:ADP-ribosyl Cyclase / Q000276:immunology*; D051997:ADP-ribosyl Cyclase 1; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015703:Antigens, CD / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping / Q000379:methods*; D008213:Lymphocyte Activation; D008280:Magnetics; D008562:Membrane Glycoproteins; D013601:T-Lymphocytes / Q000276:immunology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D017252:CD3 Complex; D008562:Membrane Glycoproteins; D036541:ADP-ribosyl Cyclase; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1