Protocol for synthesis and mechanical characterization of polyacrylamide hydrogels of varying stiffness for cell culture applications

The impact of mechanical cues on cell behavior is increasingly being recognized, rendering hydrogel platforms that mimic the extracellular matrix indispensable in in vitro cell biology research. Here, we present a protocol for the synthesis and rheological characterization of polyacrylamide (PAAm) hydrogels with varying stiffnesses, produced as large, circular, unattached gels customizable in shape and size. We outline techniques for their use in cell culture and downstream applications involving secretome or cell analysis and protein visualization by fluorescence microscopy. For complete details on the use and execution of this protocol, please refer to Cresens et al.¹.