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Summary: Assessing the strength and kinetics of molecular interactions of cells with the extracellular matrix is fundamental to understand cell adhesion processes. Given the relevance of these processes, there is a strong need for physical methods to quantitatively assess the mechanism of cell adhesion at the single-cell level, allowing discrimination of cells with different behaviors. Here we introduce single-cell acoustic force spectroscopy (scAFS), an approach that makes use of acoustic waves to exert controlled forces, up to 1 nN, to hundreds of individual cells in parallel. We demonstrate the potential of scAFS by measuring adhesion forces and kinetics of CD4+ T lymphocytes (CD4) to fibronectin. We determined that CD4 adhesion is accelerated by interleukin-7, their main regulatory cytokine, whereas CD4 binding strength remains the same. Activation of these cells likely increases their chance to bind to the vessel wall in the blood flow to infiltrate inflamed tissues and locally coordinate the immune response. : Kamsma et al. introduce single-cell acoustic force spectroscopy (scAFS), an approach using acoustic waves to exert controlled forces to hundreds of individual cells in parallel in order to measure their adhesion forces and kinetics to targets in living conditions, opening up a wide range of potential applications in research and the clinic. Keywords: acoustic force spectroscopy, adhesion strength, adhesion kinetics, CD4, T lymphocytes, fibronectin, integrin, extracellular matrix, rupture force, mechanobiology