7 aad labeling

The phenotype and efficiency of transduction of hCD34⁺ HSPCs and T lymphocytes were analyzed by Flow Cytometry (FACS Canto II Instrument and DIVA software). hCD34⁺ HSPCs were stained with PE-conjugated anti-CD90 antibody (BD PharMingen), VioBlue-conjugated anti-CD34 antibody (Miltenyi Biotec), and antigen-presenting cell (APC)-conjugated anti-CD133 antibody (Miltenyi Biotec).
PBMCs were stained with anti-CD3 monoclonal antibody before and after activation (Miltenyi Biotec). In selected experiments, T cell memory phenotype was detected by staining with monoclonal antibodies specific for CD3 (VioGreen-conjugated), CD8 (APC-Vio770-conjugated), CD45RA (PE-Vio770-conjugated), CD62L (VioBlue-conjugated), and CD95 (APC-conjugated) (Miltenyi Biotec). For both cell types, cell mortality and transduction efficiency were evaluated by 7-AAD labeling (BD PharMingen) and by the level of GFP expression, respectively.

Antibodies used include SCA1 Pacific Blue (BioLegend, 108120), CD45 PE-Cy7 (eBioscience, 25-0421-82), CD11b APC-Cy7 (BioLegend, 101226), and CD31 APC (eBioscience, 17-0311-82). SVF was prepared as for cell culture, except that following centrifugation, the resulting cell pellet was resuspended in 1 ml ACK Lysing Buffer (Invitrogen) to remove red blood cells. After inactivation with Hank’s balanced salt solution (HBSS)/2% FBS, samples were centrifuged again and resuspended in HBSS/2% FBS for antibody labeling for flow cytometry or fluorescence-activated cell sorting (FACS). Samples were analyzed on a BD LSR Fortessa analyzer or sorted on a BD FACS Aria II. Cell gating was based on comparison with unstained and fluorescence-minus-one-stained controls. Single live cells were discriminated by forward-scatter and side-scatter analysis and 7-AAD labeling (BD Bioscience), respectively. Cells were sorted into serum-free DMEM media for gene expression analysis or into complete media for cell culture. Flow cytometry standard files were analyzed using FlowJo version 10.