Shandon formal fixx 10 neutral buffered formalin

TRAP staining was performed on monocytes (n=6) after 14 and 21 days of culture in hMSC-GAG conditioned media following a protocol from the University of Rochester Medical Center (Center for Musculoskeletal Research) [60 , 61 ]. Reagents were purchased from Sigma-Aldrich (Missouri, USA). Briefly, media was aspirated from wells and cells were washed once in distilled water before fixation for 5 minutes in Shandon™ Formal-Fixx™ 10% Neutral Buffered Formalin (ThermoFisher Scientific, Massachusetts, USA). Cells were washed again in distilled water, incubated in TRAP medium for 30 minutes at 37°C, and rinsed again in distilled water. 0.02% fast green was added for 30 seconds, and finally cells were rinsed again in distilled water and air dried. Once dry, each well was imaged with a Leica DFC295 (Leica, Wetzlar, Germany) microscope to identify osteoclasts (red) and other adherent cells (green/blue) (n=6). TRAP images were compared qualitatively to images of monocytes before washing and staining.