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 behavior 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 (PAA) hydrogels with defined stiffness values that are functionalized with extracellular matrix (ECM) proteins and suitable for cell culture and imaging studies. It outlines the steps for cleaning and functionalizing glass coverslips, preparing acrylamide and bis-acrylamide mixtures of desired concentrations, initiating polymerization, and conjugating ECM 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 visualization of cellular structures and protein localization. Practical notes and troubleshooting tips are also included for maintenance of hydrogel integrity and optimization of experimental reproducibility. The workflow enables systematic investigation of how substrate stiffness regulates cell morphology, migration, cytoskeletal organization, and other mechanosensitive responses. Overall, this protocol provides a reliable framework for preparing and using tunable PAA-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. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of polyacrylamide-based hydrogels of tunable rigidities and functionalization for cell culture Support Protocol 1: Preparation of reagents for hydrogel preparation Basic Protocol 2: Staining for live-cell imaging and visualization of cellular structures and protein localization Basic Protocol 3: Chemical fixation and immunofluorescence staining for visualization of cellular structures and protein localization Support Protocol 2: Preparation of reagents and materials for cell fixation and immunostaining.