Lineage cell depletion cocktail

Primary human CD34^pos-HSPCs isolated from umbilical cord blood (CD34^pos-HSPCs) and the mononuclear cells (MNC) from whole cord blood were purchased from ALL Cells (USA). For CD34^pos-HSPCs isolation from whole cord blood, the MNCs were washed and filtered through a 100 μm cell strainer (BD Falcon) followed by lineage depleton by negative selection using a lineage cell depletion cocktail (Milteny Biotec). The lineage depleted fraction was stained with 13 markers for lineage-specfic monoclonal antibodies FITC (CD2, CD3, CD4, CD11b, CD14, CD15, CD24, CD10, CD41, CD20, CD66c, CD127, CD33) (Biolegend and BD) and Pacific blue conjugated anti-CD34 (Biolegend), for 30 min at 4 °C. Before sorting, 7-AAD was added to the tubes. Afterwards, 13Lin−CD34^pos cells were collected by the fluorescence-activated cell sorter (FACSAria, BD Biosciences, San Jose, CA, USA).
For CD34^pos-HSPCs, the cells were thawed using stem span SFEM II medium (Stem Cell Technologies, Canada) and washed 1× with the same medium. The cells were resuspended in 1 mL of medium contained 1× Stem Span Cytokine Cocktail CC100 (Stem Cell Technologies, Canada) in 24-well plate, then the cells were analyzed within 24 h.