Csu x1 spinning disk

Cells were grown in 35-mm glass-bottom dishes (MatTek) and imaged 48 h after transfection. Before imaging, medium was replaced to get rid of any debris and to ensure accuracy of volume for the PAO experiments. For kinetic PAO experiments, Arf6 KO cells expressing GFP-SNX6 were imaged for 2 min and their localization recorded. At time 0, DMSO or PAO (1 μM final) diluted in medium was added to the cells and three movies of 2 min were recorded (before 15 min, between 15 and 30 min and between 30 and 45 min). Only cells displaying at least two tubules at time 0 were kept for analysis. Live imaging was carried out using a Nikon Ti Eclipse microscope equipped with a Yokogawa CSU-X1 spinning-disk, a motorized stage and a Photometrics Evolve 512 camera (Roper Scientific) controlled with the NIS-Elements software. Movies were acquired at 37 °C and 5% CO₂ with a Tokai Hit stage-top incubator and objective heater. Fluorescence was collected with a 60 × plan apochromat immersion oil objective (NA 1.49). Images were collected at 1 × 1 binning with an exposure time of 100 ms. The number, maximum length and persistence of GFP-SNX6 tubules were analysed with ImageJ software.

Parasites were collected within a few hours following spontaneous egress from the HFF monolayers and washed in HBSS supplemented with 1% FCS. Time-lapse video microscopy was conducted in chamlide chambers (LCI Corp., Seoul, Korea) installed on an Eclipse Ti inverted confocal microscope (Nikon France Instruments, Champigny sur Marne, France) with a temperature and CO₂-controlled stage and chamber (LCI Corp., Seoul, Korea), equipped with a coolsnap HQ2 camera (Photometrics, Roper Scientific, Lisses, France) and a CSU X1 spinning disk (Yokogawa, Roper Scientific, Lisses, France). The microscope was piloted using Metamorph software (Universal Imaging Corporation, Roper Scientific, Lisses, France) and images were acquired with settings including 1 frame/second for 20 minutes, with one to two laser wavelengths depending on the experiment.