Recombinant mouse igfbp3

Mouse metatarsal culture was performed as previously described20 (link). Briefly, the middle three metatarsals were aseptically dissected from wild-type C57BL/6 mice. Bones were maintained in 500 μl α-MEM medium supplemented with 0.2% bovine serum albumin, 0.1 mM β-glycerophosphate, 50 μg ml⁻¹ ascorbic acid, 1% Pen/Strep and 0.1% Fungizone. Bone were kept individually in 24-well plates in a humidified incubator at 37 °C, 5% CO₂, for 3 days, medium was refreshed on day 2 of culture. DMSO, UNC1999 (2 μM), recombinant mouse Igf1 (100 ng ml⁻¹, R&D systems, Minneapolis, MN), recombinant mouse Igfbp3 (50 nM, R&D systems) and/or recombinant mouse Igfbp5 (50 nM, R&D systems) were added at the beginning of culture and refreshed on day 2. Hypertrophic cell size in metatarsal bones was determined taking an area of 100 μm × 100 μm at the centre of the HZ, and measuring and averaging the height of every cell within the area.

The medium used for embryo collection was HEPES (N-2-Hydroxyethylpiperazine-N’-2-ethanesulfonic acid) buffered modified synthetic human tubal fluid medium (HEPES mod-HTF; ~270–300 mOsm/kg, pH 7.4) containing 0.3 mg/mL bovine serum albumin (BSA; Sigma-Aldrich; St Louis, MO, USA). The medium used for embryo culture was potassium simplex optimized medium (KSOM; ~260 mOsm/kg, pH 7.4) containing 0.3 mg/mL BSA. All media were made up from stock solutions prepared from tissue culture grade reagents as previously described [28 (link)]. All reagents were purchased from Sigma Aldrich unless otherwise specified.
Recombinant mouse IGFBP3 (R&D Systems, Minneapolis, MN, USA) was reconstituted at a concentration of 100 μg/mL in sterile PBS. Human/mouse IGF1R neutralising antibody (IGF1R nAb; AF-305-NA; R&D Systems) was reconstituted at 0.2 mg/mL in PBS. Aliquots were prepared and stored at −80 °C. Erlotinib hydrochloride (Santa Cruz, Dallas, TX, USA) was reconstituted at a concentration of 5 mM in dimethyl sulfoxide (DMSO). Aliquots were prepared and stored at −20 °C.