High-throughput Flow-cytometry Measurement of Cellular Mechanotype Based on Rupture and Delivery of DNA Tension Probes into Cells.

Mechanical forces guide many critical cellular processes, and cellular mechanical phenotype, or mechanotype, is disrupted in many diseases. The most widely used method to quantify cellular forces is traction force microscopy, which traditionally captures forces that cells exert on their surroundings by high-resolution microscopy of cells deforming elastic substrates containing fluorescent beads, deflecting microposts, or altering the conformation of immobilized molecular tension sensors. We present here a high-throughput method to measure relative cellular traction forces using established DNA tension probes and quantified via flow cytometry. There are five overall steps involved in this method described below. First, expression and purification of the high-affinity integrin ligand echistatin fused to a DNA-linking HUH-tag; second, preparation of the DNA duplex probes containing reporters and ligand; third, surface preparation and immobilization of probes; fourth, execution of the "Rupture and Deliver" tension-gauge-tether experiment and flow readout. Finally, we will describe data analysis and interpretation of experimental results.