96 well clear bottom plate

Freshly isolated human CD14+ monocytes were resuspended in serum‐free RPMI and seeded in 96‐well clear bottom plates (Greiner Bio‐One, Stonehouse, UK) at 10⁶ cells per mL for 15 min at 37 °C in 5% CO₂. Serum‐free RPMI was replaced with 1% (vol/vol) FBS RPMI‐1640 supplemented with or without 10 µg·mL⁻¹ DQ‐BSA Red (Thermo Fisher). Cells were stimulated for 18 h with indicated treatments and imaged live using a 20× objective on an Incucyte ZOOM System (Essen BioScience, Royston, UK). Absorbance at 620 nm emission wavelength per field of view was quantified, and absorbance threshold was set using no DQ‐BSA control.

A549, CHO-WT, CHO-ΔHS or HuTu80 cells were grown as monolayers overnight at 37 °C in 96-well clear bottom plates (Greiner bio-one, Kremsmünster, Austria). For experiments blocking infection with soluble GAGs, the cell monolayers were washed three times with serum-free medium and incubated with heparin-treated virions for one hour on ice. Unbound virions were removed by washing the cells three times with serum-free medium. Medium with 1% FBS was then added and the cells were incubated for 44 h at 37 °C. Next, the cells were washed with PBS and fixed with ice-cold methanol for 10 min at −20 °C. Viral capsid proteins were stained with polyclonal rabbit antibodies diluted in PBS for 30 min at room temperature. The cells were washed twice for 15 min with PBS and then incubated with Alexa Fluor 647 goat antirabbit IgG (H+L) secondary antibody for detection of infected cells, and Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA) for visualization of cellular nuclei. After two more washes with PBS, number of transfected cells were analyzed using the Tina program of the TROPHOS (Luminy Biotech Enterprises, Marseille, France). For transduction with HAdV-F41 vector, the vector was incubated with CHO cells for two hours on ice before adding medium with 1% FBS and incubating the cells at 37 °C for 72 h. The cells were given a final PBS wash before analyzing them on the TROPHOS.

HFFs were grown to confluence in D10 in 96-well clear-bottom plates (Greiner Bio-One), then inoculated with 2 × 10⁵ tachyzoites in D3 per well, and incubated at 37°C and 5% CO₂. After 2 h, noninvaded parasites were washed away and wells were treated with vehicle (EtOH) or 0.5 mM IAA in D3 at 37°C, 5% CO₂ to degrade the mAID-3HA fusion proteins. At 24 h postinfection, the monolayers were fixed with 4% formaldehyde in PBS for 10 min, permeabilized with cold methanol for 5 min, and blocked with 10% goat serum in PBS. Parasites and vacuoles were labeled with rat anti-TgSAG1 (1:5,000) and rabbit anti-TgGRA7 (1:1,000), respectively. Next, parasites were counterstained with Alexa Fluor-dye conjugated goat secondary antibodies (1:2,000) and Hoechst 33258 dye (1:5000). Sixteen fields were imaged per replicate at 40× using a Cytation 5 plate-reading microscope running Gen5 software (BioTek Instruments). To determine the number of parasites per vacuole, at least 100 vacuoles were counted per replicate and averaged from 3 independent trials.