Hydrogel Microparticles as Force Sensors: Synthesis and Application in Phagocytosis Studies v1

We provide a protocol for the synthesis of tuneable hydrogel microparticles (HMPs), and their application to measure forces during phagocytosis by macrophages. A specific type of deformabale acrylamide-based HMP, DAAM-particles, are synthesized in batch using a membrane emulsification approach. They then are conjugated with both biologically active molecules and fluorescent labels in a one-pot functionalization procedure. Macrophages are incubated with the functionalized DAAM-particles and imaged by regular confocal microscopy. With a custom image analysis strategy, local DAAM-particle deformations can be quantified with superresolution accuracy (< 50 nm) in 3D throughout phagocytosis. Finally, normal and shear forces can be inferred from the 3D shape of DAAM-particles, revealing the spatial distribution of cellular forces. The entire protocol takes 2-3 days and requires only basic expertise in mammalian cell culture and fluorescence microscopy, and minimal specialized equipment. This protocol can be adapted for studying several other cellular processes, and thereby broadly applied in mechanobiology and immune cell biology.