HuMoSC Chemotaxis Assay with Inhibitors

HuMoSC chemotaxis was assessed using Boyden chambers with 10 μm pore polycarbonate filters. The lower chamber was filled with undiluted supernatants of control TAB or GCA-TAB cultivated for five days in MATRIGEL. A total of 30.10³ HuMoSC/well in 50 µL of 10% FBS RPMI medium were loaded in the upper chamber. In selected chambers, an antagonist of CCR2 (Calbiochem, used at 100 nM) or CCR5 (maraviroc, R&D Systems, used at 1 µM) was added. After 4.5-hour incubation, the polycarbonate membrane was stained with hematoxylin and eosin and washed before counting number of cells/field.