Designing an Attachment Assay for Keratinocytes Stimulated by Fibroblast Conditioned Media

Cell attachment involves the interaction between an individual cell with its neighbors and the extracellular matrix. These interactions are often regulated by the signaling molecules, including a variety of proteins, cytokines and growth factors. It is found in several studies that keratinocytes in the epidermis communicate with dermal fibroblasts in a bilateral paracrine model, in which they were simultaneously stimulated by the byproduct of the others. We developed this protocol with a simple, low-cost, and effective model to examine keratinocyte attachment that could be applied to examine the effects of fibroblasts on cell adhesion. For this method, we collected the conditioned media from fibroblasts and use it as a treatment for keratinocytes. Treated keratinocytes are then plated in a matrix and quantified with immunofluorescence techniques to determine the amount of attachment based on different treatment concentrations.