Cellular composition of the liquid cultures and in vivo tissues were analyzed using flow cytometry. The following antibodies and recombinant proteins were used: Sca1-Pacific blue (PB), CD34-Phycoerythrin (PE), CD48-PE, CD150-PE.Cy7, Ter119 Allophycocyanin (APC)(all Biolegend), CD117-APC, CD16/32-APC.Cy7, CD11b-PE, Gr1-APC, CD3-PE.Cy7, CD71 PE (all BD Biosciences), CD19 Alexa Fluor 700 (AF700, Life) and CD117 PerCpCy5.5 (SONY). Lineage positive cells were detected using the murine lineage detection kit (BD Biosciences) subsequently stained with streptavidin-pacific orange (PO, Fisher Scientific).
SSEA4-PE (Biolegend) and Tra-1-60 Alexa Fluor 647 (AF647, BD Biosciences) were used for regular pluripotency screenings of the iPSCs. Hematopoietic induction of iPSC was assessed using CD34-PE and CD45-BV421 antibodies (both BD Biosciences). Dead cells were excluded by using 7-aminoactinomycin-D (7AAD, Life) or 4’,6-Diamidino-2-Phenylindole (DAPI, Fisher Scientific). Flow cytometry was performed using a BD LSRII flow cytometer (BD Biosciences). For subsequent cell sorting a FACSAria (BD Biosciences) was used. Analysis of FACS data was performed using FlowJo (TreeStar).
SSEA4-PE (Biolegend) and Tra-1-60 Alexa Fluor 647 (AF647, BD Biosciences) were used for regular pluripotency screenings of the iPSCs. Hematopoietic induction of iPSC was assessed using CD34-PE and CD45-BV421 antibodies (both BD Biosciences). Dead cells were excluded by using 7-aminoactinomycin-D (7AAD, Life) or 4’,6-Diamidino-2-Phenylindole (DAPI, Fisher Scientific). Flow cytometry was performed using a BD LSRII flow cytometer (BD Biosciences). For subsequent cell sorting a FACSAria (BD Biosciences) was used. Analysis of FACS data was performed using FlowJo (TreeStar).