Ciprofloxacin hcl

Skeletal Stem Cells were cultured in alpha-MEM GlutaMax supplemented with 10% fetal bovine 1% penicillin–streptomycin (Gibco-Life Technologies, Grand Island, NY, USA), and 0.1% ciprofloxacin HCl (bioWORLD, Dublin, OH, USA). Cells were incubated under low O₂ conditions (2% atmospheric oxygen, 7.5% CO₂) for 48 h and then moved to standard conditions (5% CO₂). For information regarding reagents used refer to Supplementary Table 3.

The Click-iT EdU Imaging Kit (Invitrogen, Eugene, OR, USA) was used to assess the proliferation of skeletal stem/progenitor cells with Wnt3a treatment. Skeletal stem/progenitor cells from the three cranial sutures were isolated, pooled (as described above) and seeded at the density of 3 × 10³ cells/well in a 24-well plate. Skeletal stem/progenitor cells were cultured in alpha-MEM GlutaMax supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin (Gibco-Life Technologies, Grand Island, NY, USA), 0.1% ciprofloxacin HCl (bioWORLD, Dublin, OH, USA) and Wnt3a(50 ng/ml) (R&D Systems). Cells were incubated under low O₂ conditions (2% atmospheric oxygen, 7.5% CO₂) for 48 h and then moved to standard conditions (5% CO₂). Cells were incubated in EdU (10 μM) for 1.5 h under standard conditions. The Click-iT EdU Imaging Kit was used per the manufacturers protocol. The “Analyze Particle” tool on ImageJ imaging software was used for quantification of proliferating (GFP⁺) cells. The number of GFP⁺, proliferating cells, as well as Dapi⁺ nuclei was measured, and proliferation presented as a percentage of GFP⁺ cells/Dapi⁺ cells. For information regarding reagents used refer to Supplementary Tables 3 and  4.

Colonies were assessed as previously described^{31 (link)}. 500 freshly sorted skeletal stem/progenitor cells from pN3, CD-1 mice PF, SAG, and COR sutures and the GP were seeded into a 10 cm² plate pre-coated with 0.1% gelatin (EmbryoMax, Millipore, Burlington MA, USA) in alpha-MEM GlutaMax (supplemented with 10% fetal bovine serum 1% penicillin–streptomycin (Gibco-Life Technologies, Grand Island, NY, USA), and 0.1% ciprofloxacin HCl (bioWORLD, Dublin, OH, USA). Cells were incubated under low O₂ conditions (2% atmospheric oxygen, 7.5% CO₂) for two weeks. For evaluation of CFUs, colonies were stained with crystal violet. Cell colonies were photographed under an inverted microscope and colonies with >50 cells or more were counted. For evaluation for self-renewal ability, skeletal stem/progenitor cells were isolated by FACS and pooled from the PF, SAG, and COR sutures of pN3 CD-1 mice. 500 skeletal stem/progenitor cells were seeded into 10 cm² for two weeks as described above. After 2 weeks clones were lifted using Stem Pro Accutase (Gibco-Life Technologies, Grand Island, NY, USA) and skeletal stem/progenitor cells isolated by FACS and passaged onto 10 cm² plates as described above. For information regarding reagents used refer to Supplementary Table 3.