Apc cy7 conjugated anti c kit

Cells derived from in vitro mouse PSC hematopoietic differentiation and the in vivo PB and BM of m-NSG mice were harvested and suspended in PBS with 2% FBS. Before antibody incubation, the cells were blocked with an anti-CD16/32 antibody (eBioscience). The following antibodies were used for analysis: Alexa Fluor 700 (AF700)-conjugated anti-Flk1 (eBioscience), allophycocyanin (APC)-conjugated anti-TIE2 (eBioscience), APC-Cy7-conjugated anti-c-kit (eBioscience), AF700-conjugated anti-Lineage (eBioscience), APC-conjugated anti-Sca-1 (eBioscience), peridinin-chlorophyll protein (PerCP)-eFluor® 710-conjugated anti-CD201 (eBioscience), APC-Cy7-conjugated anti-TER119 (eBioscience), PerCP-Cy5.5-conjugated anti-CD45.1 (eBioscience), and PE-Cy7-conjugated anti-CD45.2 (eBioscience). The following antibodies were used for sorting: APC-Cy7-conjugated anti-c-kit (eBioscience), eFluor450-conjugated anti-Lineage (eBioscience), and phycoerythrin (PE)-conjugated anti-CD201 (eBioscience). Samples were measured by BD Fortessa, and cells were sorted by BD Aria II. The data were analyzed using FlowJo Version 10 software.

Bone marrow and spleen single-cell suspensions were labeled with respective antibody cocktails. For the analysis of the erythroid compartment, CD71-APCCy7 and Ter119-Pacific Blue antibodies were used (BD), and successive stages of erythroid maturation were calculated as previously described [42 (link)]. For MEP analyses, the BM cells were first treated with a 0.75% NH₄Cl solution (Sigma-Aldrich, St. Louis, MO, USA) to lyse red blood cells and labeled with a biotinylated lineage cell detection cocktail (Miltenyi Biotec) and revealed by streptavidineFluor450 (eBioscience). The cells were then labeled with PE-conjugated anti-CD34, APCCy7-conjugated anti-c-Kit, APC-conjugated anti-Sca-1, and PECy7-conjugated anti-CD16/32 (eBioscience) antibodies, and MEPs were identified as Lin−, Sca-1−, c-Kit+, CD16/32−, and CD34− [60 (link)]. At least 300,000 cells were acquired.
The cells were analyzed using a FACSCanto II and a SORP LSRII (BD) equipped with blue (488 nm), violet (405 nm), and red (638 nm) lasers. Data were analyzed with the FlowJo software.