Protocol for quantifying the adhesive strength of single fluorescent cells adhering to a non-fluorescent cell monolayer by fluidic force microscopy

Fluidic force microscopy combines the strengths of microfluidics and atomic force microscopy, allowing the analysis of cell detachment with up to several hundred nanonewton adhesion forces, without the application of chemical adhesives, which is not possible with atomic force microscopy alone. Fluidic force microscopy has been used in a variety of cell detachment studies where the cell of interest is attached to the substrate or embedded in cell monolayers. However, it might be difficult to study cells in vertical overlays with this method because they belong to adjacent vertical layers and cannot be easily identified with brightfield microscopy. Therefore, we present here a detailed protocol for the detection of adherent, fluorescent single cells on a non-fluorescent cell monolayer and for testing detachment levels using the fluidic force microscopy setup. This protocol allows the quantification of high nanonewton adhesion forces and has the potential to be applied to other types of adherent cell lines.