RosetteSep™ HLA B Cell Enrichment Cocktail

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	Human B cells were purified first with a RosetteSep HLA B cell Enrichment Cocktail (StemCell Technologies) through negative selection followed by incubation with anti-CD27–coupled magnetic beads (Miltenyi Biotec) to isolate memory B (CD27+) cells.

Protocol tips
Human B cells were purified first with a RosetteSep HLA B cell Enrichment Cocktail (StemCell Technologies) through negative selection followed by incubation with anti-CD27–coupled magnetic beads (Miltenyi Biotec) to isolate memory B (CD27+) cells.

Publication protocol

"Cell culture and reagents
Human B cells were purified first with a RosetteSep HLA B cell Enrichment Cocktail (StemCell Technologies) through negative selection followed by incubation with anti-CD27–coupled magnetic beads (Miltenyi Biotec) to isolate memory B (CD27+) cells. Naive B (CD27−) cells were obtained from the flow-through fraction. Purified human B cells at a density of 1 × 106 cells/ml were stimulated with IL-21 (100 ng/ml; Invitrogen) and anti-CD40 (1 μg/ml; R&D Systems). SKW 6.4 (SKW) human lymphoblastoid cells, 18-81 pre-B cells, 293T cells, and 3T3 cells were maintained as described (10, 19). Stable WI-L2 transfectants were maintained as described (10), and Blimp-1–estrogen ligand-binding domain (ERD) was induced by 5 μM CdCl2 (Sigma-Aldrich) and 3 μM 4-OHT (Sigma-Aldrich) treatment for 10 h. Mouse splenic B cells were purified by positive selection using B220 microbeads (Miltenyi Biotec), and the purified B cells were stimulated with LPS (2 μg/ml), goat anti-mouse F(ab′)2 (10 μg/ml; Jackson ImmunoResearch Laboratories), anti-CD40 (1 μg/ml; BD Biosciences), and IL-21 (200 ng/ml; eBioscience) at a density of 1 to 2 × 106/ml."

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