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Determination of Intracellular Compound Concentrations

Occasionally large differences in compound potency in biochemical versus cell-based assays are noted. While protein binding may account for some of this discrepancy, we have on occasion noted differences in the extent to which different compounds accumulate inside of cells or the extent to which the same compound accumulates in different cells (1).

We have developed an intracellular compound accumulation assay to measure the amount of compound inside cells over time after incubation with the compound.

Cells are incubated with compound (at a concentration below the IC50, if possible) for up to 24 hours. At varying timepoints, the cells are harvested, washed extensively with ice cold media, and frozen. The lysate is extracted with organic solvent and the compound in the lysed cells measured by LC-MS/MS.

Compound concentration can be normalized to protein content, cell number, or if the dimensions of the cell are known, to intracellular volume using the volume of an ellipsoid calculated from the cell length and width.  

(1)  Wu, C., Y. Feng, E.R. Cardenas, N..Williams, P.E. Floreancig, J.K. De Brabander, and M.G. Roth (2012) Studies toward the Unique Pederin Family Member Psymberin:  Structure−activity relationships, biochemical studies, and genetics Iientify the mode-of-action of psymberin.  J. Am. Chem. Soc. 134: 18998-19003. PMCID: PMC3504174.