Bio plex pro tgf β assay kit

AH samples were collected as described previously [29 (link)]. Levels of ATX in the AH were determined by a two-site immunoenzymatic assay with an ATX assay reagent using the AIA system (Tosoh, Tokyo, Japan). TGF-β levels in the AH were measured using a Bio-Plex Pro TGF-β assay kit (Bio-Rad Laboratories, Hercules, CA, USA) in accordance with the manufacturer’s protocol.

Protein from bone lysates (12-15 per age and sex) was harvested as previously described^{86 (link)} using Bead Mill Tubes (Thermo Fisher Scientific, Waltham, MA) containing radioimmunoprecipitation (RIPA) buffer with eComplete Mini protease inhibitor (Roche, Basil Switzerland). and homogenized using a benchtop bead mill homogenizer (Omni International, Kennesaw, GA) at 4 °C. Serum was collected from blood as previously described.^{27 (link)} Protein concentrations were quantified using the Pierce Coomassie (Bradford) Assay Kit (Thermo Fisher Scientific, Waltham, MA). Total levels of TGFβ ligand were quantified in hydrolyzed lysates by using the Bio-Plex Pro TGF-β Assay Kit (Bio-Rad, Hercules, CA) on a Bio-Plex 200 (Bio-Rad, Hercules, CA). TGFβ ligand levels were normalized to each sample’s original total protein concentration.