Murine progenitor enrichment cocktail

The bone marrow from femur and tibia were suspended in cold PBS with 2% FBS and passed through a 70 μm filter. Filtered bone marrow cells were suspended in PBS with 2% FBS and 0.1 g/L phenol red and then enriched for lineage negative (Lin−) cells using the SpinSep system (Stem Cell Technologies, Vancouver, BC, Canada). The cells were incubated with a murine progenitor enrichment cocktail (Stem Cell Technologies) on ice for 30 min, washed, and then incubated with dense particles on ice for 20 min. The cells were then centrifuged at 1200g for 10 min, and the cells at the density medium/PBS interface were collected.
Enriched MSCs were seeded onto culture plates at a density of 0.1 × 10⁶ cells/cm² in α-MEM containing 100 units/ml penicillin (Gibco) and 100 μg/ml streptomycin (Gibco). The media were changed after 72 h and adherent cells were maintained in culture with twice weekly media changes.
To induce osteogenic differentiation, BMSCs were treated with 100 nM dexamethasone, 10 mM β-glycerophosphate disodium and 50 μg/ml ascorbic acid.
BMSCs were treated in vitro with tubastatin A (Sigma) in DMSO at 8 μM for 21 days. Vehicle controls were treated with culture medium with DMSO only.

Femur bone marrow was suspended in PBS and passed through a filter. The filtered bone marrow cells were suspended in PBS containing 2% FBS, incubated with a murine progenitor enrichment cocktail (Stem Cell Technologies, Vancouver, BC, Canada) on ice for 30 min, washed, and then seeded onto culture plates at a density of 1 × 10⁵ cells/cm² in α-MEM containing 10% FBS, 100 units/mL penicillin (Gibco BRL, Rockville, MD, USA) and 100 μg/mL streptomycin (Gibco). The media was changed after 3 days, and adherent cells were cultured with twice weekly media changes. BMSCs at passages <3 were used in the current study.
BMSCs were treated in vitro with 50 and 100 ng/ml recombinant mouse CCL12 (R&D Systems, Minneapolis, MN).

BMSC was isolated as previously described [26 ]. The bone marrow from femur and tibia were suspended in cold PBS and passed through a 70 μm filter. The supernatants were kept to be used in ELISA. Filtered bone marrow cells were suspended in PBS with 2% FBS and 0.1 g/L phenol red and then enriched for lineage negative (Lin−) cells using the SpinSep system (Stem Cell Technologies, Vancouver, BC, Canada). The cells were incubated with a murine progenitor enrichment cocktail (Stem Cell Technologies) on ice for 30 min, washed, and then incubated with dense particles on ice for 20 min. The cells were then centrifuged at 1200 g for 10 min, and the cells at the density medium/PBS interface were collected.
Enriched BMSC were seeded onto culture plates at a density of 0.1 × 10⁶ cells/cm² in α-MEM containing 100 units/ml penicillin (Gibco BRL, Rockville, MD, USA) and 100 μg/ml streptomycin (Gibco). The media were changed after 72 h and adherent cells were maintained in culture with twice weekly media changes.