Identification of antigen-specific B cells by concurrent monitoring of intracellular Ca2+ mobilization and antigen binding with microwell array chip system equipped with a CCD imager.
B cells are very heterogeneous, consisting of more than 10(9) B-cell clones with distinct specificities for antigens in each individual. To identify single B cells with antigen specificity, we have been developing cell microarray technology using microwell array chips whose microwells each capture a single B cell. Using microwell array chips, we detected antigen-specific B cells by monitoring antigen-induced intracellular Ca2+ mobilization with a CCD scanner (MAC-CCD system) or the binding of fluorescence-labeled antigen to cells with a confocal laser scanner. We retrieved target cells from the chip, cloned immunoglobulin genes, and produced antigen-specific antibodies. However, these methods present some difficulties: the former technique could not detect cells whose frequency was less than 0.05% and the latter one took a long time to identify the objective cells although it could detect cells at a frequency of 0.01%. Here, we have combined the advantages of these two methods. Monitoring antigen-induced intracellular Ca2+ mobilizations and the binding of fluorescence-labeled antigens simultaneously with a MAC-CCD system enabled us to detect rapidly, antigen-specific B cells whose frequency was less than 0.01% with high efficiency. Our system provides a superior screening system for antigen-specific B cells and extends the horizons of multiparameter single-cell analysis in heterogeneous cell populations.
Kinoshita
Koshi
K
Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Japan.
Ozawa
Tatsuhiko
T
Tajiri
Kazuto
K
Kadowaki
Shinichi
S
Kishi
Hiroyuki
H
Muraguchi
Atsushi
A
D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism; D000941:Antigens / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D020013:Calcium Signaling; D002452:Cell Count; D000939:Epitopes / Q000276:immunology*; D005456:Fluorescent Dyes / Q000378:metabolism; D019044:Image Cytometry / Q000295:instrumentation*; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D046888:Tissue Array Analysis / Q000295:instrumentation* / Q000379:methods
D000906:Antibodies; D000941:Antigens; D000939:Epitopes; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D002118:Calcium