Assaying Surface Expression of Chemosensory Receptors in Heterologous Cells

Summary

Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Abstract

The vivid world of odors is recognized by the sense of olfaction. Olfaction in mice is mediated by a repertoire of about 1200 G Protein Coupled Receptors (GPCRs) ¹ that are postulated to bind volatile odorant molecules and converting the extracellular signal into an intracellular signal by coupling with G protein Gαolf. Binding of the odorants to the receptors is thought to follow a combinatorial rule, that is, one odorant may bind several receptors and one receptor may bind several odorants to varying degrees ². Biochemical, signaling and ligand binding studies have been conveniently carried out for most GPCRs using heterologous cells. However use of heterologous cells for study of odorant receptors, was precluded for a long time since on transfection they failed to export to the surface. Saito et al have demonstrated single membrane pass Receptor Transporting Protein (RTP) family chaperones show enhanced expression in the olfactory sensory neurons and act as chaperones to traffic odorant receptors to the surface in heterologous cells, when co transfected together ³. To carry out biochemical assays for receptors using heterologous cells, one must first determine if the receptor shows robust surface expression in the cell line. This can be assayed by overexpressing the receptors with the chaperone RTP1S followed by live cell staining to fluorescently label the extracellular domain or a tag in the extracellular domain exclusively. Here we demonstrate a protocol to carry out live cell staining that can be used to detect odorant receptors on the surface of HEK293T cells conveniently. In addition, it may also be used to assay for surface expression of other chemosensory receptors or GPCRs.

Protocol

1. Procedure:

The procedure comprises of three parts completed over a total time of 3 days: transferring cells, transfection and immunocytochemistry, one step carried out per day. Transfer and transfection of cells over the first two days must be carried out in sterile conditions in a laminar flow chamber.

Day 1: Transfer of HEK293T cells for surface expression assay.

1. Transfer medium (M10): Mix minimum essential medium supplemented with 10% (by volume) fetal bovine serum in sterile condition.
2. Grow cells to a desired confluency in a 100mm culture plate, depending on the number of plates required ....

Discussion

Each step must be carried out carefully to ensure prominent and distinctive surface staining. The entire staining process must be carried out in cold (on ice), and the tray on which cover slips are laid must be cooled prior to use to ensure cells stay alive. Moreover fixation is done at the end of the surface staining process. This is in sharp contrast with internal staining where fixation precedes staining to permeabilize the cell membrane to primary and secondary antibodies. Time of exposure of cover slips to air must .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cover slip 22 X 22 mm (#1)	Thermo Fisher Scientific, Inc.	72200-11
Fetal bovine serum	GIBCO, by Life Technologies	16000
Lipofectamine 2000	Invitrogen	11668-019
Minimal essential medium	Sigma-Aldrich	M4655	With L glutamate, Earle’s salt and bicarbonate
Mowiol	Calbiochem	475904
Paraformaldehyde	EMS	19208
Hanks Balanced Salt Solution (HBSS) 1X	GIBCO, by Life Technologies	14025	With calcium chloride and magnesium chloride
HEPES Buffer Solution	GIBCO, by Life Technologies	15630
Phosphate Buffer Saline (PBS)	Cellgro	21-040-CV	Without calcium and magnesium
Poly D Lysine Hydrobromide	Sigma-Aldrich	P7280
Sodium Azide	Electron Microscopy Sciences	SX0299-1
Tissue culture dish 35 X 10 mm	Falcon BD	353801
Trypsin-EDTA	GIBCO, by Life Technologies	25300	0.05%