In this work, we developed and optimized a high-throughput assay to study pSmad pathways using immunofluorescence (IF) as an alternative to Western blot. We aimed to overcome the technical challenges usually faced in the classical IF assay in image acquisition, analysis, and quantification.

We used C2C12 cells as a cellular model. The cells were stimulated with BMP-2 and TGF-β1 that were delivered either in solution (soluble) or via a biomaterial presenting the growth factor (GF), that is in a "matrix-bound" manner. Image acquisition parameters, analysis methods, and quantification of pSmads using IF were optimized for cells cultured on two types of supports: on bare glass and on a biomimetic coating made by self-assembly of the biopolymers hyaluronic acid and poly(L-lysine), which was crosslinked and then loaded with the GFs.

We performed high-content kinetic studies of pSmad expression for cells cultured in 96-well microplates in response to soluble and matrix-bound BMP-2 and TGF-β1. The detection limit of the IF-based assay was found to be similar to Abbreviations: ALP, alkaline phosphatase; bBMP-2, matrix-bound BMP-2 loaded in the biomimetic films; BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptors; bTGF-β1, matrix-bound TGF-β1 loaded in the biomimetic films; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, 1:1 Dulbecco's Modified Eagle Medium; DMSO, dimethyl sulfoxide; EC50, half-maximal effective concentration; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HA, hyaluronic acid; HEK293, human embryonic kidney cells; IC50, half-maximal inhibitory concentration; IF, immunofluorescence; PEI, polyethylenimine); PFA, paraformaldehyde; PLL, poly(L-lysine) hydrobromide; pSmad, phosphorylated Smad; sBMP-2, soluble BMP-2; sTGF-β1, soluble TGF-β1; Sulfo-NHS, N-hydroxysulfosuccinimide sodium salt; TCPS, tissue culture polystyrene; TGF-β, transforming growth factor; TGF-βR, transforming growth factor receptor.