Data quality is not affected by cell suspension density
When determining the potencies of drugs in single-cell assays it is a major concern that the employed assay is affected by factors such as the density of the applied cell suspension. It is envisaged that a high density of cells may lead to titration of test compound molecules (by receptor-binding and adsorption) by cells in the vicinity of the targeted cell. This would cause a rightward shift in the concentration-response relationship, and consequently, incorrectly increased IC50 values. We have tested whether IC50 values determined with the QPatch 16 automated patch-clamp system are dependent on the density of the applied cell suspension. We assume that cell density determines the number of cells present in the extracellular flow channel.
It is predicted that increasing the cell suspension density may reduce the time it takes to position a cell on the patch-clamp site. The cell positioning step is accomplished by suction. We tested the effect of the cell suspension density using a wide range of densities (from 0.5 to 16 million cells per ml) on the cell positioning time. For the tests we used two expression systems: (1) CHO cells expressing hERG potassium channels, and (2) HEK cells expressing Nav1.2a sodium channels. We used test compounds with very different oilwater partition coefficient (log(P)): astemizole and verapamil with log(P) values of 6.43 and 3.45, respectively, (source: www.syrres.com/esc/physdemo) for hERG assays, and tetrodotoxin (TTX) with log(P) of -6.21 for the Nav1.2a assay.