Human foreskin fibroblast (HFFs) cultures show U-I cells after incubation with <i>Toxoplasma</i>-β-lactamase strain.

<p>HFFs were plated at near-confluence and incubated with a RH-mCherry-β-lactamase strain at an MOI of 0.5 for 2 hours prior to loading for 30 minutes with the detection substrate (CCF2-AM). Live images were obtained using a confocal microscope. Green and blue fluorescence represent the uncleaved and cleaved CCF2-AM substrate, respectively; red represents mCherry expression by the parasites. (a) and (b) are representative color-merged images from the same experiment. White arrows indicate cells with substrate cleavage (blue) but no detectable parasite. Empty arrowheads indicate infected cells with substrate cleavage. Right panels are enlarged views of boxed regions in the left panels. Scale bars = 10 µm. (c) Flow cytometry analysis of fully confluent cultures incubated with a RH-mCherry-β-lactamase strain at an MOI of 0.5 for 2 hours prior to substrate loading. Panels from left to right represent contour plots of unstained, uninfected HFF cultures used to draw gates for blue⁺, mCherry⁻ and blue⁺, mCherry⁺ cells; gating applied to the contour plot of HFFs incubated with the RH-mCherry-β-lactamase strain and loaded with CCF2-AM; and the histogram of the prior panel comparing the mean fluorescence intensity of blue of the U-I cells (upper-left gate) compared to infected cells (upper-right gate).