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. 2011 Jun 9;6(7):953-60.
doi: 10.1038/nprot.2011.344.

Magnetic-based Purification of Untouched Mouse Germinal Center B Cells for Ex Vivo Manipulation and Biochemical Analysis

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Magnetic-based Purification of Untouched Mouse Germinal Center B Cells for Ex Vivo Manipulation and Biochemical Analysis

Matthew H Cato et al. Nat Protoc. .
Free PMC article

Abstract

Detailed biochemical analysis of unmanipulated germinal center (GC) B cells has not been achieved. Previously, we designed and used a simple, economical and new magnetic bead separation scheme for the purification of 'untouched' mature GC and non-GC B cells from the spleens of immunized mice and reported the first biochemical assessment of the signaling cascades that contribute to cyclin D stability and GC B cell proliferation. Here we provide a detailed protocol for the method we used, which involves preparing single-cell suspension from the spleens of immunized mice, followed by labeling of nontarget cells with biotinylated antibodies specific for CD43, CD11c and IgD (for GC enrichment) or GL7 (for non-GC enrichment); these steps are followed by cell depletion using standard magnetic bead technology. This protocol can yield GC and non-GC B cells with purities exceeding 90%. The sorting process can be carried out in ∼1 h and provides a population of GC B cells of sufficient purity and quantity to allow ex vivo manipulation, including biochemical and genetic analysis as well as cell culture.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Flowchart of Ab labeling and magnetic sorting steps for sorting non-GC and GC B cells.
Figure 2
Figure 2
Representative flow cytometry plots. (ab) A successful sort (a), and an unsuccessful sort (b). Presorted total splenocytes are shown in black (a, upper left). Blue and red gates indicate GC and non-GC populations, respectively, as indicated by GL7 and Fas (CD95) staining (a, upper left). Post-sort purity for GCs (a, upper right) and non-GCs (a, lower left) are indicated as the percentage of total events in the single-cell gate. For non-GCs, B220 and IgD staining confirm that GL7 Fas cells are mature B2 cells (a, lower right). Frequency of CD11c+ dendritic cells and CD138+ plasma cells are indicated as percentage of GL7 Fas non-GCs (b, top) from an unsuccessful GC sort (b, bottom).

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