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Updated: May 27, 2026
Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils
Published on: November 16, 2015
Multiparametric flow cytometry for identification and fluorescence activated cell sorting of five distinct B-cell
Malene Krag Kjeldsen1, Martin Perez-Andres, Alexander Schmitz
1Department of Haematology, Aalborg Hospital, Aarhus University Hospital, Aalborg, Denmark.
Insights
Researchers developed a fast method to isolate five distinct B-cell subpopulations from human tonsils. This technique enables detailed molecular analysis of B-cell differentiation and function.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Human tonsils are a rich source of various B-cell subpopulations crucial for immune responses.
- Understanding B-cell differentiation requires methods to isolate distinct subpopulations for molecular analysis.
- Previous methods may lack the speed or specificity needed for simultaneous isolation of multiple B-cell types.
Purpose of the Study:
- To establish a robust and rapid procedure for isolating distinct B-cell subpopulations from human tonsils.
- To provide a basis for subsequent in-depth molecular and functional analyses of these purified B-cell subsets.
Main Methods:
- Isolation of five distinct B-cell subpopulations (naive B cells, centroblasts, centrocytes, memory B cells, plasmablasts) using fluorescence-activated cell sorting (FACS) based on surface marker expression.
- Verification of immunophenotypic identity using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR).
- Analysis of proliferation marker (MKI-67) and B-cell differentiation markers (BACH2, BCL6, PAX5, IRF4, PRDM1, XBP1).
Main Results:
- Successfully purified five distinct B-cell subpopulations from fresh human tonsils.
- Confirmed the identity and purity of sorted subpopulations using qRT-PCR.
- Distinguished large and small centroblasts, noting differences in proliferation and gene expression of germinal center markers (BCL6, BACH2).
Conclusions:
- A detailed and rapid procedure for the simultaneous isolation of up to five distinct maturation-associated B-cell subpopulations from human tonsils has been established.
- This method facilitates the study of B-cell heterogeneity and differentiation pathways.
- The findings provide a foundation for future molecular investigations into B-cell biology and immune responses.
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.
