35 mm fluorodishes

Pre-cleaned microscope slides 24 × 75 × 1 mm, coverslip glass 24 × 40, and Nunclon Sphera plates were purchased from Fisher Scientific (Waltham, MA). High tolerance dishes (P35G-0.170-14-C) were obtained from MatTek Corporation (Ashland, MA). 35 mm fluorodishes were obtained from World Precision Instruments (Sarasota, Fl). Silicone culture inserts for self-insertion were purchased from Idibi (Grafelfing, Germany).

MEFs seeded at a density of 40 000 cells per ml onto 35 mm-FluoroDishes (World Precision Instruments) were transfected with either mtRFP or mtYFP. Twenty-four hours after transfection, MEFs were supplemented with 10 mM HEPES and transferred to an Axio Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany), the incubation chamber was pre-heated to 37 °C. For each experiment, images were captured at 10 s intervals using an Axio Cam MR3 (Carl Zeiss). In some experiments, actin drugs (0.25 μm JASP or 0.5 μM CYTD, both diluted in DMSO) were directly applied to the culture medium 10 min after starting the recordings, indicated by time point 0 min on the abscissae of Figures 2e and g.

ThRK13 cells were seeded onto 35 mm fluorodishes (World Precision Instruments, FL, USA) in normal growth media (see Cell Culture). Next day, cells were incubated for 2.5 hr (35°C, 5% CO₂) with Opti‐MEM/200 μM 2‐NBDG in DMSO/100 μM Hoescht 33342 (Thermo Scientific, MA, USA). Cells were treated with Opti‐MEM/Calcein AM 2.5/100 μM Hoescht 33342 and incubated for 1 hr (35°C, 5% CO₂). Cells were then washed and media replaced with Ham's F12 media (Gibco, Thermo Scientific, MA, USA). Dishes were sealed and cells imaged using the Nikon N‐SIM Microscope system mounted with a 37°C stage, fitted with an iXon + 897 EMCCD camera (Andor Technology, Ulster, UK).
Images were assessed quantitatively using Image J. Gray level values were plotted along 3 μm long linear intercepts that were drawn radially in a direction from the center of each cell profile. As the lines crossed the cell periphery orthogonally they were positions so that 1.5 μm of the line was on the inside of the cell and 1.5 μm was the outside. For each cell profile, five intercepts were measured at angles selected systematic random but only recorded when the cell periphery was adjacent to host cytoplasm and not when other parasites were present. Each local gray value was expressed as a fraction of the density range for that intercept ranging from 0 to 1 (mean density value, Figure 11).