Abstract
Larvae of the anhydrobiotic midge Polypedilum vanderplanki show the extreme desiccation tolerance.
Recently, Pv11 cell line was established from Polypedilum vanderplanki embryos. Pv11 cells exhibit the
desiccation tolerance when treated with trehalose before desiccation. During trehalose treatment, the
expression level of various genes drastically changes. It is suggested that trehalose stimulation is transmitted
to cells by signaling activation. Nevertheless, it is unclear which second messengers work during trehalose
treatment. We can evaluate the second messenger behavior by live-cell imaging using sensor proteins. However,
the live-cell imaging for Pv11 cells is difficult because Pv11 cells are floating cells. Here, we introduce the
live-cell calcium imaging method in Pv11 cells by immobilization with Biocompatible Anchor for cell
Membrane (BAM). We established the Ca2+ indicator stable expressing Pv11 cell line (Pv11-GCaMP6f). Then,
we immobilized Pv11-GCaMP6f with BAM and added the calcium ionophore by using perfusion system.
Finally, we succeeded to develop the method for real-time Ca2+ imaging at a single cell level.
