EasySep™ Human Cord Blood CD34 Positive Selection Kit II

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	CD34+ cells were isolated using EasySep™ human cord blood CD34 positive selection kit II (cat# 17896, Stemcell Technologies, Vancouver, BC, Canada) and stored at −150 °C.

Protocol tips
CD34+ cells were isolated using EasySep™ human cord blood CD34 positive selection kit II (cat# 17896, Stemcell Technologies, Vancouver, BC, Canada) and stored at −150 °C.

Publication protocol

Anonymized human UCB samples were collected from healthy new-borns of both sexes at University Hospital Basel. Relevant ethical regulations were followed, according to the guidelines of the local Basel ethics committees (vote 13/2007V, S-112/2010, EKNZ2015/335). UCB cells were processed by density gradient centrifugation, and CD34+ cells were isolated using EasySep™ human cord blood CD34 positive selection kit II (cat# 17896, Stemcell Technologies, Vancouver, BC, Canada) and stored at −150 °C. Upon thawing, CD34+ cells were stained with the following antibodies: CD34 (APC conjugate, clone: 581, 1 : 100 dilution, BD Biosciences), CD38 (PEcy7, HB7, 1 : 200, BioLegend), CD45RA (FITC, HI100, 1 : 100, BioLegend), CD90 (PE, 5E10, 1 : 50, BD Biosciences) and CD49f (PEcy5, GoH3, 1 : 50, BD Biosciences) for 30 minutes at 4 °C. Cells were then washed and resuspended in PBS + 2% FCS with 1 μg ml−1 7-aminoactinomycin D (7-AAD, ThermoFisher Scientific). HSPCs, phenotypically defined as CD34+CD38−CD45RA−CD90+CD49f+, were sorted using a BD™ FACSAria III with 100 μm nozzle, purity mode and sorting purities ≥90%. Gates and thresholds were set according to fluorescence minus one (FMO) controls.

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