Cell immobilization for live imaging
We used two methods to immobilize cells for live imaging. For both methods, we prepared a single slide at a time and viewed it immediately. The first method relied on the pressure exerted on small volume samples under a cover slip. Cells were first concentrated by centrifugation to 2-5 x 106 cells ml-1. 6 μl of sample was applied to the slide, and immediately overlayed with a 22 X 22 mm cover slip. The resulting pressure frequently results in cell immobilization, which could be verified by monitoring ciliary beating on the cell surface at the same time that cell viability could be monitored by observing the periodic contraction of the contractile vacuole, both in the DIC channel. For the second method, cells were pelleted and resuspended in a thermoreversible gel on ice, whose viscosity increases as it warms to room temperature. Here, cells at the same high density were mixed well with CyGELTM (ab109204, ABCAM) (the mix ratio optimized for each experiment) and immediately mounted with a cover slip. This approach results in many cells being effectively immobilized in the thin gel. For both methods, we carefully watched for proliferation of large cytosolic vesicles. Growing cells have large food vacuoles derived from the oral apparatus. No new food vacuoles form in immobilized cells, so any substantial increase in the number of such vesicles is likely to represent autophagosome formation as part of a stress response. We rejected any cells showing such an increase.