μ slide 2 well chambered coverslip

For live cell microscopy, infected monolayers were prepared on a μ-Slide 2-well chambered coverslip (Ibidi) (0.5x10⁶ cells/well infected at a 1:1 ratio). The μ-Slide was placed onto the stage of a confocal microscope (LSM 880 AxioObserver – Zeiss) which was set at 41°C, 5% CO₂. Nile Red (200 μg/ml in acetone) was added to each well. Fluorescence imaging was performed using continuous z-stack acquisition encompassing all cell-containing focal planes. Images were acquired for both differential interference contrast (DIC) and fluorescence. Imaging was performed using either a 40x objective (voxel size 0.42 micrometer (μm) x 0.42 μm x 1-2 μm) or a 63x objective (voxel size 0.26 μm x 0.26 μm x 0.59 μm). Each field was imaged for a maximum of 15 minutes to minimise thermal damage to the cells. Staining of free live sporozoites with Nile Red at different concentrations (0.5 μg/ml to 100 μg/ml) did not show significant differences in ABR presence, proving that Nile Red has no deleterious effect in sporozoites and does not affect RBant. loss.

Bacterial conjugation was visualized over time on a Celldiscoverer 7 live cell imaging microscope (Zeiss). For these experiments, the GFP-DD donor strain was mixed with 36_WT-expressing recipients that constitutively express dTomato. Overnight cultures of donor and recipient bacteria were washed in PBS, mixed in a 1:1 ratio and 8 μl was spotted onto a 1 cm² 2% agarose (w/v) pad supplemented with M9 salts and 0.4% glucose (w/v). The pad was inverted into a μ-Slide 2-well chambered coverslip (Ibidi, 80286). The sample was maintained at 37 °C throughout live imaging. Images were acquired every 10 min for 3.5 h and processed using Zen 2.3 (Blue Version, Zeiss).