TY - JOUR
T1 - Multiparametric flow cytometry for identification and fluorescence activated cell sorting of five distinct B-cell subpopulations in normal tonsil tissue
AU - Kjeldsen, Malene Krag
AU - Perez-Andres, Martin
AU - Schmitz, Alexander
AU - Johansen, Preben
AU - Boegsted, Martin
AU - Nyegaard, Mette
AU - Gaihede, Michael
AU - Bukh, Anne
AU - Johnsen, Hans E
AU - Orfao, Alberto
AU - Dybkær, Karen
PY - 2011
Y1 - 2011
N2 - 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.
AB - 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.
U2 - 10.1309/AJCPDQNP2U5DZHVV
DO - 10.1309/AJCPDQNP2U5DZHVV
M3 - Journal article
SN - 0002-9173
VL - 136
SP - 960
EP - 969
JO - American Journal of Clinical Pathology
JF - American Journal of Clinical Pathology
ER -