Picro sirius red assay

Osteoblasts were cultured from neonatal α9KO (n = 10) and α9WT (n = 7) C57BL/6J mice at day 1–4 according to the protocol of [27 (link),28 (link)]. The neonatals were a kind gift of Dr. Claudia Dames (Institute of Medical Immunology, Charite - Universitätsmedizin Berlin, Germany). In brief, calvaria and long bones were incubated in 1 mg/mL collagenase (Capricorn Scientific, Ebsdorfergrund, Germany) and 4 mM ethylene diamine tetra acetic acid (EDTA) at 37 °C and centrifuged for 5 min at 1.500 g at 4 °C. Isolates were seeded in 24 well plates for 4 d at 37 °C and 5% CO₂ and passaged with 0.005% trypsin (Gibco, Life Technologies, Carlsbad, USA). Cells of passage 2 (13.441 cells/cm²) were used for osteogenic differentiation in α-minimum essential medium (MEM, Gibco) containing 10% fetal bovine serum (FBS, PAN Biotech, Aidenbach, Germany), 10⁻⁷M dexamethasone (Sigma, St. Louis, MO, USA), 50 pg/mL sodium L-ascorbate (Sigma), 2 × 10⁻³ M β-glycero phosphate hydrate (Sigma), and gentamicin/amphotericin (Gibco) for 7 d. Then, mineralization was determined using the Senso- Lyte pNPP alkaline phosphatase assay (AnaSpec, Fermont, CA, USA) and collagen deposition was analyzed by a Picro-Sirius-Red assay (Abcam, Cambridge, UK). Subsequent histomorphometry calculated the area of positive collagen type 1 and 3 staining.