C11440 orca flash 4

Where MitoTracker staining was used, cells were stained with 200 nM MitoTracker Red CMXRos (Invitrogen) in complete medium for 30 min prior to fixation. The cells were fixed with 4% w/v formaldehyde in PBS, permeabilised with 0.2% v/v Triton X-100 for 15 min or 1 min, and blocked with 2% w/v bovine serum albumin for 1 h. The cells were stained with 1:100 rabbit anti-C1QBP (Abcam #ab270032) overnight at 4°C followed by a mixture of 1:1000 goat anti-rabbit 488 (Invitrogen #A11008) and 5 μg/mL DAPI for 1 h at room temperature. Where cells were infected and MitoTracker staining was not used, the cells were additionally stained with 1:500 mouse anti-ROP1 (Abnova #MAB17504) overnight at 4°C followed by 1:1000 goat anti-mouse 594 (Invitrogen #A11005) for 1 h at room temperature. Images were acquired on a Nikon Ti-E inverted widefield fluorescence microscope with a Nikon CFI APO TIRF 100x/1.49 objective and Hamamatsu C11440 ORCA Flash 4.0 camera running NIS Elements (Nikon).

Videos were obtained using a digital CMOS camera (HAMAMATSU, C11440/ORCA-Flash 4.0) mounted to Nikon TE-300 inverted microscope with an LED light engine (Lumencor SpectraX). A heated platform (Tokai Hit, TPi-SQX) was used to image the MPS at 37 °C. For electrical stimulation, a pulse generator (ION OPTIX Myopacer Field Simulator) was used. 20 V biphasic rectangular pulses (20 ms) were applied via 1.5″ long blunt stainless-steel needles (Vita Needle M937) inserted into the media inlet and outlet pipette tips. Calcium traces were recorded at 100 fps with 4 × 4 binning using the cyan LED for excitation. A 1 mM isoproterenol stock was prepared freshly from isoproterenol hydrochloride (I0260; TCI Chemicals) powder right before the drug response experiments, sterile filtered and diluted to 1 µM. 100 µL of drug-containing media were applied to each chip via the inlet pipette tip 30 min before recording.

60,000 BMDMs per well of a Falcon black-walled, clear-bottom 96-well plate were stimulated with IFNγ for 24 h as described above. The plates were infected with parasite lines at an MOI of 0.3 or 3 at the same time as propidium iodide (Invitrogen) was added to a concentration of 5 μg/mL. Images of each well were acquired every 30 minutes between 1 h and 12 h post-infection on a Nikon Ti-E inverted widefield fluorescence microscope maintained at 37°C with a Nikon CFI Plan Fluor 4x/0.13 objective and Hamamatsu C11440 ORCA Flash 4.0 camera running NIS Elements (Nikon). After 12 h, Triton X-100 was added to a concentration of 1% v/v to fully permeabilise the cells and a final image was captured of each well. In each image, the total fluorescence signal was measured. The percentage of propidium iodide uptake in each well at each timepoint was calculated by subtracting the first measurement at 1 hpi to remove background fluorescence signal and normalising the total fluorescence intensity following full permeabilisation to 100% uptake. The mean propidium iodide uptake across three technical replicates for each strain was taken to represent each biological replicate. Differences between strains were tested by paired two-sided t-test with Benjamini-Hochberg adjustment.