Table 4.

Troubleshooting

Problem	Possible causes	Solution
Few or no cells under microscope (Step 10A (xvi)) or during flow cytometry (Step 10B (xvii)).	Cells lost during the experiment	Plate cells evenly and sparsely when transferring cells for transfection. Use gentle techniques during staining and washing. Dense spots of cells tend to peel off during immunocytochemistry.
No cell-surface expression (Step 10A (xvi) or 10B (xvii) of the Procedure)	Staining did not work due to factors such as bad antibody solutions	Verify cell-surface expression of positive controls.
	Transfection did not work	Verify BFP expression in live-cell staining or GFP expression in flow cytometry.
	The OR of interest does not have cell-surface expression	Verify OR protein expression by permeabilized staining or Western Blot.
Unusually low firefly and Renilla luciferase measurements (Steps 10C (vii) and 10C (xi) of the Procedure)	Cells were lost during the experiment	Plate cells evenly and sparsely when transferring cells for transfection and use gentle techniques during transfection, washing, and stimulation. Dense spots of cells tend to peel off during medium change.
	Cells died due to odorant toxicity	Luciferase measurements may be normal for lower concentrations of the same odorant. Use lower concentrations.
	Cells died due to defective 96-well plates	Observe the cells carefully before transfection.