Slide 1 luer

In vitro magnetic retention assays under flow conditions were performed in a modified channel slide (µ-Slide I Luer, 0.4 mm height, ibidi). This modification allowed the two-magnet system, specially designed for this assay, to be placed on the chamber and produce a homogeneous strong magnetic gradient on both sides of the channel. Jurkat or murine primary T cells (10⁷ cells/ml) were incubated or left untreated with different doses of MNPs (50, 100 and 150 µg Fe/ml) for 2 h in standard conditions, washed with PBS and stained with calcein-AM (ThermoFisher) for 20 min at 37 °C in PBS with 0.5% FBS. Cells were then diluted in PBS with 0.5% FBS (2.5 × 10⁵ cells/ml) until use. Flow chamber set-up was mounted on an Olympus Inverted Microscope model IX71 connected to an Imaging Station cell^R and in standard culture conditions (37 °C 5% CO₂, 90% relative humidity). PBS with 0.5% FBS was infused at high flow rate to fill the system. Cells were infused at 0.5 dyne/cm² (≈ 100 μl/min). Images were recorded every 1 s for 4 min. After the first 60 s, the two-magnet system was placed into the flow chamber and its effect recorded for the remaining time. IMARIS Software (Bitplane) was used to generate the movies and for cell motility tracking. Displacement in Y-axes (magnetic-field direction) was analysed.

FL-VWF protein (20 µg/ml) was perfused through a flow chamber slide (µ-slide I luer, Cat# 80176, Ibidi, Germany), with shear stress of 5 dyn/cm² for 2 min with the same concentration of bovine serum albumin (BSA) (Sangon Biotech) used as a control. E. hellem spores (10⁵/ml) were then perfused through the channel for 1 min. The channels were washed with PBS, and then fixed with 4% paraformaldehyde. The VWF “strings” along the channel were visualized under a fluorescent microscope after incubation with anti-VWF IgG (ab6994, Abcam, USA) followed by Alexa 594-labeled secondary antibody. The E. hellem spores were visualized by Calcofluor-white (CFW) (Sigma-Aldrich), a specific dye for chitin on the microsporidia spore surface (Luna et al., 1995 (link)).

Larvae were washed twice using artificial seawater. The larvae were washed in artificial seawater supplemented with 25 mM EGTA, then lysed for 1 h in Triton X-100 1% in the same buffer on ice. We could observe the appearance of a gel containing bristles. The gel was washed twice in 10 mM Tris-HCl, 2.5 mM MgCl₂, 0.5 mM, CaCl₂ pH 7.6. 50 µL were left in the sample at the last wash, and the gel was digested with 5 U of DNase I (Thermo Fischer Scientific) for 2.5 h at 37 °C with 360 rpm agitation. Next, the digest was supplemented with 100 µL of artificial seawater and digested with 200 µL of 2.5 µg/µL Liberase Enzyme Blend (Sigma-Aldrich) for 1 h at 50 °C. The bristles were washed three times in artificial seawater. The isolated bristles were mounted on µ Slide I Luer (Ibidi). The refractive index tomograms of isolated bristles were obtained by Nanolive 3D Cell Explorer, and raw data were deposited at 10.5281/zenodo.10207240.