Simultaneous, real-time imaging of intracellular calcium and cellular traction force production.
Cells can sense and respond to different types of mechanical stimuli that can lead to changes in rate of cell division, cell orientation, cell motility, and gene expression. There is rapidly growing interest in understanding how these processes are regulated by mechano-chemical signaling mechanisms. The movement offish epithelial keratocytes is regulated by the activation of stretch-activated calcium channels, which allow cells to trigger retraction of the rear cell margin, when forward movement is impeded. We have developed a new assay that permits imaging of intracellular calcium concentration simultaneously with the detection of traction forces generated by moving keratocytes. The assay consists of a thin sheet of gelatin embedded with a surface layer of small fluorescent marker beads, on which cells can move. The elastic properties of the gelatin substrata can be reproducibly varied over a wide range and are stable for long periods, while submerged beneath culture medium. Gelatin substrata are thin, transparent, and highly elastic, allowing real-time detection of changes in traction force production that are associated with transient increases in intracellular calcium and that occur in response to mechanical stretching.
Doyle
Andrew D
AD
University of Connecticut, Storrs 06269, USA.
Lee
Juliet
J
D000180:Acrylic Resins; D000818:Animals; D002118:Calcium / Q000032:analysis*; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D004548:Elasticity; D019573:Epithelium, Corneal / Q000502:physiology*; D017276:Friction; D005780:Gelatin; D005782:Gels; D007089:Image Enhancement; D008422:Materials Testing; D008856:Microscopy, Fluorescence / Q000379:methods*; D011036:Poecilia / Q000502:physiology*; D012212:Rheology / Q000379:methods; D020034:Silicone Gels; D013314:Stress, Mechanical; D013696:Temperature; D014159:Transducers; D014783:Viscosity
D000180:Acrylic Resins; D005782:Gels; D020034:Silicone Gels; C016680:polyacrylamide gels; D005780:Gelatin; D002118:Calcium