Description
Background: At present, B cell differentiation and development are recognized as very complex. High speed multi-parameter flow cytometry (MFC) and cell sorting (FACS) enables fast and sensitive identification and characterization of normal and malignant B-cell hierarchy.Hypothesis and aims: We hypothesize that B-cell subpopulations identified by MFC have distinct gene expression profiles reflecting their global functions. The aims are to use MFC, sort identified B-cell subpopulations for gene expression profiling (GEP) for a better understanding of normal function and differentiation.
Methods: From homogenized human tonsil tissue, isolated mononuclear cells are subjected to MFC and FACS utilizing a multicolor fluorescence single tube panel (CD3/CD10/CD20/CD27/CD38/CD44/CD45/CXCR4), to identify and isolate distinct B-cell subpopulations for morphological inspection, global and single gene expression profiling.
Results: 5 B-cell subsets from human tonsil, namely naïve, centroblast, centrocyte, memory, and plasmablast have been MFC identified, and FACS sorted based on their immunophenotype. The cellular identity of the subpopulations was verified at the gene expression level using microarray and qRT-PCR gene expression profiling based on the used discrimination CD markers as well as transcription factors KI-67 (proliferation) and (BACH2, BCL6, PAX5, IRF4, PRDM1, XBP1 (B-cell differentiation markers).
Conclusion: With a combination of surface marker expressed antigens and gene expression analysis of B cell subsets a strong methodology is provided to generate improved insights into the B-cell biology and thereby also development of B-cell malignancies.
| Period | 25 Sept 2010 |
|---|---|
| Event title | 10th Euroconference on Clinical Cell Analysis (ESCCA 2010) |
| Event type | Conference |
| Location | Valencia, SpainShow on map |