Measuring cellular force using DNA-based tension probes: from ensemble to single-molecule studies

Understanding how cells sense and respond to mechanical forces is crucial for many biological processes, including adhesion, migration, differentiation and immune activation. In this protocol, we describe two advanced DNA-based tension probes, the reversible shearing DNA-based tension probe (RSDTP) and ForceChrono probe, which provide powerful tools for studying mechanotransduction in living cells. RSDTPs enable dynamic quantification of forces ranging from 4 to 60 pN, offering the advantage of reversibility without ligand depletion, making them ideal for ensemble force measurements across populations of cells. ForceChrono probes not only measure the magnitude of force but also capture its duration and loading rate, providing essential insights into the temporal dynamics of single-molecule force transmission. We detail the fundamental principles, design strategies and step-by-step procedures for synthesizing, purifying and applying these probes, including surface preparation, cell experiments, image acquisition and data analysis. In addition, we describe the computational tools for image analysis. Together, these probes enable a detailed analysis of cellular mechanobiology, with applications in integrin mechanobiology and cell adhesion biology. This protocol is suitable for researchers with a background in cell biology, molecular biology, surface chemistry, optical imaging and data analysis and can be completed by a graduate student in 3-4 days.