Abstract
Recently, discrimination of numerous odors with high sensitivity grows in demand. Olfactory system is the pattern
recognition of the activation of olfactory receptors. We can mimic olfactory system by evaluating olfactory receptor’s response. Insect olfactory receptors are heterotetramers consisting of one olfactory receptor (OR) and the olfactory receptor co-receptor (Orco). They form a ligand-gated cation ion channel. Sf9 cells can grow at room temperature without CO2 gas and can produce high amount of proteins. Thus, Sf9 cells are commonly used to express olfactory receptors. One way to evaluate the response to odorants is live-cell calcium imaging. However, the live-cell imaging for Sf9 cells are difficult because their adherence capability onto a culture plate is low relatively. Here, we introduce the live-cell calcium imaging method in Sf9 cells by immobilization with low melting point agarose (LMPA). Or47a and Orco from Drosophila melanogaster were cloned and co-expressed in Sf9 cells with a calcium indicator protein GCaMP6f. We dropped an OR-expressing Sf9 cells solution to a plate and covered it with LMPA to immobilize. After addition of a ligand solution to immobilized Sf9 cells, we succeeded to observe the OR response in the same Sf9 cells without any toxicity by LMPA.
