Protocol for measuring weak cellular traction forces using well-controlled ultra-soft polyacrylamide gels

Traction force microscopy (TFM) is a popular technique for studying cellular stresses; however, the reproducible fabrication of ultrasoft substrates for the reliable detec-tion of weak cellular stresses (below 100 Pa) remains a challenge. Here, we describe a simple in vitro TFM protocol using such ultrasoft protein-coated polyacrylamide gels and wide-field fluorescence microscopy. We complement the protocol with open-source and in-house scripts for data analysis for the easy quantification of trac-tion stresses, which is demonstrated here using peripheral blood mononuclear cells.