Using tunable hydrogel microparticles to measure cellular forces

Force generation is an integral part of cellular behavior. It plays a crucial role in cell adhesion, migration and division. Mechanical forces are also essential in cell-to-cell interactions, including the widespread interactions involving immune cells. Accurately measuring these forces remains a major challenge, yet it is essential for understanding the mechanobiological mechanisms driving these interactions. Here, we describe a methodology in which deformable and tunable hydrogel microparticles are used to quantify cellular forces. A specific type of acrylamide-based tunable hydrogel microparticles, deformable poly-acrylamide co-acrylic acid microparticles (DAAM-particles), are synthesized in batch using a membrane emulsification approach and conjugated with both biologically active molecules and fluorescent labels through a one-pot functionalization procedure. Cells are then incubated with functionalized DAAM-particles and imaged by confocal microscopy. With a custom image-analysis strategy, local microparticle deformations can be quantified with super-resolution accuracy (<50 nm). Elasticity theory calculations allow for the inference of normal and shear forces, revealing the direction and spatial distribution of cellular forces. The tunability of DAAM-particles enables their adaptation for investigating numerous cellular processes, making them a valuable tool for understanding mechanobiology. The entire protocol takes 2-3 d, requires only basic expertise in mammalian cell culture and fluorescence microscopy and utilizes less specialized equipment and facilities compared with other available techniques. As an example, we demonstrate how this methodology can reveal actin-based force generation during phagocytosis by macrophages.