Ckit cd117 apc

The following directly labeled anti-mouse monoclonal antibodies were used for BM cell phenotypical analysis: CD90.2-APC and CD45-PE/Cy7 for lymphoid progenitors, CD61-APC and CD41-FITC for megakaryocytic population, CD71-PE and Ter119-FITC for erythroid precursors, CD11b-PE and Gr1-FITC for granulocytes/monocytes progenitors, Lineage Cocktail (CD3, Gr1, CD11b, CD45R, Ter119)-FITC, Sca1-PE, cKit (CD117)-APC for hematopoietic stem cells, all purchased from BioLegend (BioLegend, San Diego, CA, USA).
The phenotypical analysis of splenocytes was performed using the following anti-mouse antibodies: CD4-PE/Cy5, CD8a-PE (BioLegend) for helper and cytotoxic T cells, CD19 (BioLegend) for B cells, CD11c-PE, I-Ab-FITC, and TLR4 (CD284)-PE/Cy7 (all from BioLegend) for dendritic cells (DCs), and NK1.1-FITC (BioLegend) for NK cells. To detect proliferative cells, Ki67-eFluor660 (eBioscience, San Diego. USA) was used.
Single-cell suspensions of splenocytes or BM cells were incubated with the fluorescently labeled antibodies in PBS containing 1% BSA, at 4°C for 20 min for cell surface staining. For intracellular staining (Ki67), cells were permeabilized using the Foxp3 Fix/Perm Buffer (eBioscience), according to the manufacturer’s instructions. Measurements were performed with a FACSCalibur flow cytometer as described above.

Bone marrow cells (0.5 × 10⁶) were stained with B220-FITC (Becton Dickinson (BD) Pharmingen, Franklin Lakes, NJ, USA), IgM-PE (Southern Biotechnology Associates, Inc., Birmingham, AL, USA), c-kit (CD117)-APC (Biolegend, San Diego, CA, USA), CD25-APC (Biolegend), CD19-Brilliant Violet 421 (Biolegend), and CD93-PE-Cy7 (Biolegend). Splenocytes (0.5 × 10⁶) were stained with B220-FITC (BD Pharmingen), CD21-FITC (BD Bioscience), IgM-PE (Southern Biotechnology Associates, Inc.), CD93-APC (eBioscience, Vienna, Austria), CD19-Brilliant Violet 421 (Biolegend), and CD23-PE-Cy7 (eBioscience). All cells were analyzed in a FACS Canto II (BD) and data were further processed in Flow Jo version 10.0.4 (Three Star, Inc., Ashland, USA). All analyses started with a singlet gate, thereafter a lymphocyte gate and gates for indicated populations. Results are presented as absolute number of cells of the different populations.