Fibroblast chemotaxis is implicated in many physiological processes, including wound healing and morphogenesis. We present a novel assay for cherootaxis of fibroblasts (and other slow-moving tissue cells) in a direct- viewing chamber containing a physiologically relevant three-dimensional fibrin or collagen gel in which long-lasting, spatially continuous gradients have been sustained for at least 24 h, long enough for significant fibroblast migration. This combination of features is not available in any alternative assay of comparable setup simplicity. Using a putative fibroblast chemotactic factor, the fibronectin peptide GRGDSP, we measured human foreskin fibroblast alignment in the direction along the gradient, which followed a biphasic dependence on GRGDSP concentration with an optimal concentration of about 10 nM. Time-lapse video microscopy revealed that cell migration was up the soluble GRGDSP gradient, confirming positive cherootaxis to GRGDSP and rejecting the possibility of dominant haptotaxis down the soluble GRGDSP gradient, that is, up a putative gradient of integrin-mediated adhesion induced by the soluble GRGDSP gradient.
Bibliographical noteFunding Information:
This work has been supported by NIH P01-GM50150-03S1 (R.T.T.) and an NSF Research Training Grant BIR-9413241 (D.M.K.). The technical assistance of Jodi Balik, Cheryl Wotus, and Cynthia Coulter is also gratefully acknowledged.
- Cherootaxis assay
- Collagen gel
- Fibrin gel
- Fibroblast cherootaxis
- Fibronectin peptide