title: Multiparametric flow cytometry for identification and fluorescence activated cell sorting of five distinct B-cell subpopulations in normal tonsil tissue.
abstract: The purpose of this study was to establish a procedure capable of isolating distinct B-cell subpopulations from human tonsils as a basis for subsequent molecular analyses. Overall, 5 distinct B-cell subpopulations were purified from fresh tonsils based on their fluorescence surface marker expression: naive B cells, centroblasts, centrocytes, memory B cells, and plasmablasts. The immunophenotypic identity of the subpopulations was verified by quantitative real-time reverse transcriptase-polymerase chain reaction using the proliferation marker MKI-67 and 6 B-cell-associated differentiation markers (BACH2, BCL6, PAX5, IRF4, PRDM1, and XBP1). Furthermore, within the centroblast compartment, large and small centroblasts could be distinguished and large centroblasts were shown to proliferate with a morphologic appearance of a "centroblast"-like cell but with lower gene expression of the germinal center markers BCL6 and BACH2 vs small centroblasts. This study has established a detailed and fast procedure for simultaneous sorting of up to 5 distinct maturation-associated B-cell subpopulations from human tonsils.
authors.affiliation: Department of Haematology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark.
authors.forename: Malene Krag
authors.initials: MK
authors.lastname: Kjeldsen
authors.forename: Martin
authors.initials: M
authors.lastname: Perez-Andres
authors.forename: Alexander
authors.initials: A
authors.lastname: Schmitz
authors.forename: Preben
authors.initials: P
authors.lastname: Johansen
authors.forename: Martin
authors.initials: M
authors.lastname: Boegsted
authors.forename: Mette
authors.initials: M
authors.lastname: Nyegaard
authors.forename: Michael
authors.initials: M
authors.lastname: Gaihede
authors.forename: Anne
authors.initials: A
authors.lastname: Bukh
authors.forename: Hans E
authors.initials: HE
authors.lastname: Johnsen
authors.forename: Alberto
authors.initials: A
authors.lastname: Orfao
authors.forename: Karen
authors.initials: K
authors.lastname: Dybkaer
mesh_terms: D000293:Adolescent; D000328:Adult; D001402:B-Lymphocytes / Q000166:cytology*; D050976:Basic-Leucine Zipper Transcription Factors / Q000032:analysis; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D002648:Child; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D018858:Germinal Center / Q000166:cytology; D006801:Humans; D008875:Middle Aged; D014066:Palatine Tonsil / Q000166:cytology*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D012097:Repressor Proteins / Q000032:analysis
chemical_list: C491123:BACH2 protein, human; C411984:BCOR protein, human; D050976:Basic-Leucine Zipper Transcription Factors; D011518:Proto-Oncogene Proteins; D012097:Repressor Proteins