Traction Cytometry Assay: A Method to Quantify Traction Force Exerted by Cancer Cells on ECM
Transkript
– Cells generate mechanical forces that act on an extracellular matrix. This force is called traction force. Cells exert traction forces to perform various tasks such as migration and proliferation.
Metastatic cancer cells exert greater traction forces than healthy cells; therefore, we perform a traction cytometry assay to measure the traction forces exerted by cells on elastic substrates. Begin by seeding the desired amount of colon cancer cells on a polyacrylamide hydrogel, a gel that mimics ECM in vitro. The gel comprises fibronectin proteins, which help cells to adhere to the surface, and fluorescent beads, which act as a marker for positional changes.
Incubate the cells for the desired period. The cells adhere to the gel and exert the traction force, which causes displacement of the fluorescent beads. Now, place the plate under a fluorescent microscope and take an image of the displaced fluorescent beads. Next, add the desired amount of trypsin to the plate to detach the cells and analyze the traction force exerted by the cells using appropriate software. In the following protocol, we will perform a traction force microscopy assay to analyze cellular traction of human colon cancer cells.
– To perform the traction force microscopy assay, first, warm 0.25% trypsin-EDTA 10% SDS solution to 37 degrees Celsius in a water bath for 10 minutes before traction experiment start. Remove one gel at a time from the cell culture incubator and place on the microscope stage. After finding a single cell in the field of view, remove the Petri dish lid and take a phase contrast image of the cell.
Next, switch the imaging mode to fluorescence and select the appropriate filter. Don't move the microscope stage or sample during this time. Then, take an image of the fluorescent beads displaced by cellular traction. Now, add 1 milliliter of Trypsin SDS solution to the Petri dish to detach the cell from the gel. Take a reference image of the beads after the cell is removed.
