Fig. 4.

Human intestinal enteroids (HIEs) are less responsive than Vero cells to purified Clostridioides difficile toxin A: HIEs derived from jejunum were transduced with the LifeAct-Ruby sensor, which labels F-actin with red fluorescent protein. Cell rounding was visualized by live-cell microscopy on a Nikon TiE with a ×20 Plan Apo (NA 0.75) differential interference contrast objective, using a SPECTRA X LED light source and ORCA-Flash 4.0 sCMOS camera (scale bar, 50 µm). A: representative images of LifeAct-Ruby HIEs over time (1–16 h) after exposure to Fluorobrite DMEM medium alone or purified TcdA (List Biologicals) in Fluorobrite DMEM. Insets indicate that significant rounding occurs with TcdA as low as 0.01 μg/mL (10 ng/mL). Similar to the C. difficile strains, cell rounding in response to purified toxins occurs at later time points. B: FIJI (formerly ImageJ) software was used to define cell membranes (as denoted by actin labeling) and cell diameter over time. C: resulting curves were assessed for the area under the curve (AUC), which demonstrates decreased cell diameter with TcdA. *P < 0.05, 1-way ANOVA; n = 3 independent experiments, n = 6/experiment.