Cytoskeleton and Focal Adhesion Organization Assay: An Immunofluorescence-based Method to Study Cell Adhesion and Spreading on Substrates

- Substrate morphology greatly influences cancer cells' behavior and metastatic responses. A dynamic network of actin cytoskeleton provides structural support to cells and mediates cellular movement. Large membrane-associated protein complexes called focal adhesions connect the cytoskeleton with the extracellular matrix, facilitating cell-spreading.

To begin staining, take a cancer cell culture, and treat them with paraformaldehyde to fix and permeabilize the cells. Now, incubate the cells with a signal enhancer reagent containing proteins to prepare the cells to reduce background staining. Add anti-vinculin primary antibodies that bind to vinculin, one of the proteins within the focal adhesion complex.

Next, incubate the sample with a fluorescent dye conjugated secondary antibody targeting the primary antibody. Finally, treat the cells with fluorescent tagged phalloidin conjugates that selectively bind to actin filaments of the cytoskeleton. Observe the cells under a confocal laser scanning microscope to study their fluorescent signals.

Cells with well-spread morphology exhibit defined actin fibers and present distinct and elongated vinculin-containing focal adhesions. Conversely,