Protocol for the Preparation of Polyacrylamide Hydrogels with Tunable Stiffness and Their Application in Cell Culture, Live-Cell Imaging and Fixed-Cell Immunofluorescence

Tissue stiffness changes markedly in many pathological conditions, such as cancer and fibrosis, influencing cellular behaviour and function. To investigate these effects in vitro, it is necessary to culture cells on substrates whose mechanical properties can be tuned to mimic both normal and diseased tissue environments. This protocol provides a detailed, reproducible procedure for preparing polyacrylamide (PA) hydrogels with defined stiffness values that are functionalised with extracellular matrix proteins and suitable for cell culture and imaging studies. It outlines the steps for cleaning and functionalising glass coverslips, preparing acrylamide and bis-acrylamide mixtures of desired concentrations, initiating polymerisation, and conjugating extracellular matrix proteins to support cell adhesion. The protocol further describes the process of seeding cells onto the hydrogels, followed by live-cell imaging, chemical fixation, and staining for visualisation of cellular structures and protein localisation. Practical notes and troubleshooting tips are also included for maintenance of hydrogel integrity and optimisation of experimental reproducibility. The workflow enables systematic investigation of how substrate stiffness regulates cell morphology, migration, cytoskeletal organisation, and other mechanosensitive responses. Overall, this protocol provides a reliable framework for preparing and using tunable polyacrylamide-based hydrogels in studies of cell–matrix interactions, mechanotransduction, and disease-related changes in tissue mechanics. It is adaptable to a wide range of cell types and compatible with various imaging modalities, offering a versatile platform for mechanobiology research