Apc cy7 conjugated streptavidin

To avoid non-specific antibody binding, cells were treated with anti-mouse CD16/32 Fc binding blocker (eBioscience, San Diego, CA) for 10 minutes at 4°C. After one PBS wash, cells were stained with PE-conjugated anti-mouse CD45 antibody, PE-Cy7-conjugated anti-mouse c-Kit antibody, FITC-conjugated anti-mouse Sca-1 antibody and APC-Cy7-conjugated anti-mouse Mac-1 (CD11b) antibody (all from Biolegend, San Diego, CA). Pacific Blue-conjugated anti-mouse F4/80 antibody (Biolegend) was used to remove macrophages from CD45(+) c-Kit(+) cells in muscle tissue surrounding femurs. To further characterize CD45(+) c-Kit(+) Sca-1(+) cells, biotin-conjugated anti-mouse CD150 antibody (Biolegend), anti-mouse CD34 antibody (Biolegend) and anti-mouse EPCR antibody (Biolegend) were used for staining and were then detected using V500-conjugated streptavidin (BD Bioscience). To analyze lineage marker expression, cells were stained with biotin-conjugated anti-mouse Ter119, Gr-1, CD4, CD8, B220 and Mac-1 or F4/80 antibodies, followed by detection using APC-Cy7-conjugated streptavidin (all from Biolegend). Cells sorted with BD FACS Aria (BD Bioscience) were collected into RNAlater^® (Life Technologies, Carlsbad, CA) for gene expression analysis. Cells used for morphological analysis were sorted into medium containing 10% FBS and stained using May-Grünwald Giemsa staining.

BM cells were suspended in FACS buffer (2.5% BSA; 0.5 mM EDTA, in PBS) following red cell-lysis with 0.8% NH₄Cl buffer. Phenotypic surface markers were labeled using PE-conjugated anti-CD3/B220/Ter119, FITC anti-CD11b, PerCP-Cy5.5 anti-Ly6C, APC anti-Ly6G or CD11b, and Biotin anti-CD115 antibodies and APC Cy7 conjugated streptavidin (Biolegend). Cells were stained with 7AAD (BD Bioscience, UK) to assess cell death. For hematopoietic stem cell (HSC) analysis, PE-conjugated lineage cocktail and PE-conjugated isotype antibodies were used alongside FITC anti-Sca-1 and APC anti-CD117 antibodies. Data were acquired using a FACS Canto flow cytometer and analyzed using FlowJo Software (Tree Star Inc, OR, USA, version 8.8.7) and populations were gated using isotype and fluorescence minus one controls.