Flow cytometric analysis of cellular F-MTX staining and its displacement by MTX. After intracellular saturation of DHFR with 2 μM F-MTX for 8 hours, cells were incubated for 3 hours with increasing concentrations of MTX to initiate cellular F-MTX displacement. Residual F-MTX labeling was then determined by flow cytometry. A representative graph is shown from a total of 3 independent experiments. Actual cell numbers obtained for each MTX concentration in the various transfectants are shown in the inset.