RosetteSep™ Human B Cell Enrichment Cocktail

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	CLL cells were negatively selected from fresh blood samples using the RosetteSepTM B cell enrichment cocktail (StemCell Technologies, Grenoble, France) and density gradient centrifugation (Ficoll®Paque Plus, GE Healthcare Life sciences, Velizy-Villacoublay, France).

Protocol tips
CLL cells were negatively selected from fresh blood samples using the RosetteSepTM B cell enrichment cocktail (StemCell Technologies, Grenoble, France) and density gradient centrifugation (Ficoll®Paque Plus, GE Healthcare Life sciences, Velizy-Villacoublay, France).

Publication protocol

Peripheral lymphocytes were isolated from 65 untreated CLL patients and from 36 healthy blood donors (buffy coats obtained from the Etablissement Français du Sang Grand Est, Strasbourg, France). All subjects gave written informed consent for this study, which was approved by the institutional review board of the Strasbourg University Hospitals and all experiments were performed in accordance with relevant guidelines and regulations. CLL cells were negatively selected from fresh blood samples using the RosetteSepTM B cell enrichment cocktail (StemCell Technologies, Grenoble, France) and density gradient centrifugation (Ficoll®Paque Plus, GE Healthcare Life sciences, Velizy-Villacoublay, France). IGHV gene mutation status and ZAP70 expression were evaluated for each patient following established protocols48,49. Cytogenetic abnormalities were identified by metaphase analysis and fluorescence in situ hybridization (FISH) using a panel of probes as previously reported50. Total (CD19+) or naïve (CD19+, CD27+, IgM+) B cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy blood donors using a negative selection kit (Human naïve B cell isolation kit, Human B cell isolation kit, StemcellTM Technologies, Grenoble, France) after density gradient centrifugation (Ficoll®Paque Plus, GE Healthcare Life sciences, Velizy-Villacoublay, France). The cell purity was then controlled by flow cytometry on a Cytomics FC500 System (Beckman-Coulter, Fullerton, CA) using CD19+ or CD19+/CD27− staining (Beckman Coulter, Villepinte, France). CLL B cell purity was assessed after CD19+/CD5+ staining (Beckman Coulter, Villepinte, France) and ranged from 90% to 99% (median 97%). Cell differentiation was studied after anti-CD38 and anti-CD138 stainings (Beckman Coulter, Villepinte, France) at days 0 and 6.

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