Cellview plates

Neutrophils (5 × 10⁵) were seeded in 35 mm CELLview plates (Greiner Bio-One, Americana, SP, Brazil) and allowed to adhere for 30 min at 37 °C. Afterwards, non-adherent cells were washed out and T. gondii tachyzoites from RH strain were added at a parasites:neutrophil ratio of 5:1 in 800 µL of RPMI. Propidium iodide was added to the dish at a final concentration of 3 µg/mL, in order to stain NET as well as dead neutrophils and parasites over time. Parasites were allowed to settle for 15 min before recording began. Spontaneous death of parasites in culture medium in the absence of neutrophils was also evaluated as a control condition. Time-lapse analysis was performed by sequential acquisition along several hours with a time interval of 30 s, using a Zeiss Axio Observer Z1 microscope. Images were acquired by an HMR Axiocam monochrome camera operated by Axiovision software version 3.2 (Zeiss, Germany). The red signal from propidium iodide was acquired by Colibri illumination system using a 590 nm LED with Zeiss fluorescence filter 50. Temperature and focus were maintained along time-lapse analysis using a Temperature Control module and a Zeiss Definitive Focus device, respectively. Images were processed in ImageJ 1.52 (National Institutes of Health, Bethesda, MD, USA).

To assess intracellular uptake of NPs, 26,000 C28-I2 cells/cm² were seeded on Cellview™ plates (Greiner Bio-one, Kremsmünster, Austria), using DMEM/F-12 supplemented with 10% fetal bovine serum (FBS, Biowest, Nuaillé, FR), 0.2 mM ascorbic-2-phosphate (Sigma-Aldrich, The Netherlands), and 100 µg/mL penicillin and streptomycin (Gibco, ThermoFischer, Waltham, MA, USA). Cells were cultured for 24 h in a humidified incubator at 37 °C and 5% CO₂. Subsequently, 40 µg/mL of all NPs were added to the cells and incubated for an additional 24 h in un-supplemented DMEM/F-12. Cells were then fixed with formalin for 20 min, followed by 0.2% PBS-Triton for 20 min, and blocked with 5% BSA/PBS for 30 min. Subsequently, they were stained with 2.5 µg/mL of phalloidin-TRITC (Millipore-Sigma, Burlington, MA, USA) and 100 ng/mL of DAPI (Millipore-Sigma, Burlington, MA, USA) for 1 h. Between each step, cells were washed three times with 0.05% Tween/PBS. Images were acquired using a Leica SP8X confocal microscope (Leica Microsystems, Wetzlar, Germany) with a 63×/1.4 oil immersion objective. Image processing and analysis were performed with Fiji software, version 1.50 (National Institutes of Health, Bethesda, MA, USA).