Pe conjugated anti mouse sca1 and apc conjugated anti mouse c kit

Whole bone marrow cells were isolated from femurs and tibias by grinding the bones in hematopoietic stem cell (HSC) buffer [Hanks’ balanced salt solution (HBSS) with Ca2⁺ and Mg2⁺, 5% fetal bovine serum, 2 mM EDTA]. Red blood cells (RBCs) were lysed using ACK lysing buffer (Lonza, Basel, Switzerland). The total cell number was counted with a Coulter counter (Beckman Coulter Inc., Brea, CA). Three million cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated lineage cocktail (anti-mouse CD3, CD4, CD8, B220, CD11b, Gr-1 and Ter-119 antibodies), PE conjugated anti-mouse Sca1 and APC conjugated anti-mouse c-Kit (eBioscience). Dead cells were excluded by staining with 7-AAD (BD Pharmigen). Data were collected from 1 million single cells by FACSCanto (BD Biosciences) and analyzed by FlowJo software.
For colony-forming cell (CFC) assays, 2 × 10⁴ RBC-depleted whole bone marrow cells were plated in MethoCult GF 3434 (StemCell Technologies) and colonies were counted 7 days later.

Whole bone marrow cells were isolated from femurs and tibias by grinding the bones in hematopoietic stem cell (HSC) buffer [Hanks’ balanced salt solution (HBSS) with Ca2⁺ and Mg2⁺, 5% fetal bovine serum, 2 mM EDTA]. Red blood cells (RBCs) were lysed using ACK lysing buffer (Lonza, Basel, Switzerland). Cells were blocked with a rat anti-mouse CD16/32 antibody (BD Pharmingen) and stained with PE-Cy5 conjugated lineage cocktail containing anti-mouse CD3, CD4, CD8, B220, CD11b, Gr-1 and Ter-119 antibodies (eBioscience). Cells were stained with either PE conjugated anti-mouse Sca1 and APC conjugated anti-mouse c-Kit (eBioscience) or with CD27 conjugated to APC (Thermo Fisher) and CD201 conjugated to PE (eBioscience). Dead cells were excluded by staining with 7-AAD (BD Pharmingen). Data were collected from 1 million single cells by FACSCanto (BD Biosciences) and analyzed by FlowJo software.