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.