A robust diffusion-based gradient generator for dynamic cell assays

Abstract

This manuscript describes a new method to generate purely diffusive chemical gradients that can be modified in time. The device is simple in its design and easy to use, which makes it amenable to study biological processes that involve static or dynamic chemical gradients such as chemotaxis. We describe the theory underlying the convection-free gradient generator, illustrate the design to implement the theory, and present a protocol to align multiple layers of double sided tape and laminates to fabricate the device. Using this device, a population of mammalian cells was exposed to different concentrations of a toxin within a concentration gradient in a 48 h experiment. Cells were probed dynamically by cycling the gradient on and off, and cell response was monitored using time-lapse fluorescence microscopy. The experiment and results illustrate the type of applications involving dynamic cell behavior that can be targeted with this type of gradient generator.