Complete Kit for Human Whole Blood CD34+ Cells

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	CD34+ cells were purified from fresh peripheral-blood mononuclear cells using a complete kit for Human whole-blood CD34+ cells and cultured in StemCell specific medium supplemented with StemSpan CC110 Cytokine cocktail from StemCell (100x).

Protocol tips
CD34+ cells were purified from fresh peripheral-blood mononuclear cells using a complete kit for Human whole-blood CD34+ cells and cultured in StemCell specific medium supplemented with StemSpan CC110 Cytokine cocktail from StemCell (100x).

Publication protocol

"EML1 cells14 (kind gift from Dr Schickwann Tsai at the University of Utah, UT, USA) were grown in IMDM (Iscove Modified Dulbecco’s Medium) supplemented with 20% heat-inactivated horse serum and 15% BHK/MKL cell-conditioned medium. SUP-B15 and REH cell lines (from DSMZ) were grown respectively in RPMI or McCoy’s 5A supplemented with 20% heat-inactivated horse serum. Lineage minus cells (Lin−) were purified from the bone marrow of C57Bl/6 mice using a negative selection Mouse Hematopoietic progenitor kit (StemCell, Vancouver, Canada; #13056A) and then cultured in RPMI 10% FCS, SCF (100 ng/ml), IL6 and IL3 (10 ng/ml).

CD34+ cells were purified from fresh peripheral-blood mononuclear cells using a complete kit for Human whole-blood CD34+ cells and cultured in StemCell specific medium supplemented with StemSpan CC110 Cytokine cocktail from StemCell (100x).

EML1, Lin− and CD34+ cells were all induced to differentiate into B-cells using the following protocol: OP9 stromal cells (ATCC, Georgetown University in Washington, Washington, DC, USA) were plated in six-well plates in Alpha Minimum Essential Medium (α-MEM; Invitrogen, Carlsbad, CA, USA) with 20% of fetal bovine serum (GIBCO, Carlsbad, CA, USA). When they reached confluency, 50 000 EML, Lin− or CD34+ cells were plated on top of the stromal cells in their specific culture medium (see above) plus supplements (IL7 10 ng/ml; FLT3L 80 ng/ml; β-mercaptoethanol, 50 μM). IFNβ (from Santa Cruz Biotechnology, Dallas, TX, USA) was added 100 U/ml in culture at B0 of differentiation whereas Bryostatin was added 3 days before the cells were collected (B3) at the concentration of 10 nM (Santa Cruz Biotechnology). Rapamaycin and AZD8055 (Santa Cruz Biotechnology) were added at day B0 of B-cell differentiation induction at the concentration of 10 nM. Medium was changed and all cytokines were added every 3 days when requested by the protocol. All treatments were repeated in at least three independent replicates."

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