Tween 20

Before dissection, the surface of medaka bodies was washed three times with PBS containing 0.2% Tween® 20 (molecular biology grade; Promega, Madison, WI, USA) to avoid outer bacterial contamination. After washing, whole intestinal tissues were extracted, and intestinal tracts were filled with PBS and squeezed to extract intestinal contents. From each individual medaka's intestinal content, bacterial genomic DNA was extracted separately (not normalized) using the QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. The concentration of each DNA sample was set to 5 ng/μL using nuclease-free water for the PCR reaction (not pooled).

Supernatants were collected for Luminex analysis using the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Customized 7-plex Panel (Merck Millipore) to measure the following targets: IL-1α, IL-1β, IL-1RA, IL-6, IL-8, IL-10, and TNF-α. Harvested supernatants and standards were incubated with fluorescent-coded magnetic beads pre-coated with respective antibodies in a black 96-well clear-bottom plate overnight at 4°C. After incubation, plates were washed five times with wash buffer (PBS with 1% BSA (Capricorn Scientific) and 0.05% Tween-20 (Promega)). Sample-antibody-bead complexes were incubated with biotinylated detection antibodies for 1-h, and subsequently, Streptavidin-PE was added and incubated for another 30 min. Plates were washed five times again, before sample-antibody-bead complexes were re-suspended in sheath fluid for acquisition on the FLEXMAP® 3D (Luminex) using xPONENT® 4.0 (Luminex) software. Data analysis was done on Bio-Plex Manager^TM 6.1.1 (Bio-Rad). Standard curves were generated with a five-parameter logistic algorithm, reporting values for both mean florescence intensity (MFI) and concentration data.

As described previously [7 (link)], lysis buffer containing 0.5 mg/mL protease K (Amresco, US), 50 mM KCl (Sigma), 10 mM Tris-Cl, pH 8.0 (Invitrogen), 2.5 mM MgCl₂ (Sigma), 0.45% IGEPAL (Sigma), 0.45% Tween-20 (Promega, US), and DEPC-treated water (Promega) was used for the total RNA extraction. Briefly, 200 µL of lysis buffer was added to each well of 96-well plates that contained PIEs and was incubated for 30 min at 37 °C. Then, 100 µL of the PIE–lysis buffer mixture was transferred to a 96-well PCR plate, followed by incubation in a PCR cycler with the following parameters: 30 min at 65 °C and 2 min at 98 °C, and the samples were then cooled down to 4 °C. The RNA concentration was measured by NanoDrop2000c and adjusted to the same level. A Qiagen one-step RT-PCR kit was used for RVC detection using the primers, probe, and protocol described previously [18 (link)]. The Ct values from the RT-PCR were converted to FFU/mL based on a standard curve generated using the infectious titer of the cell culture-adapted RVC Cowden. Our calculations demonstrated that the mRNA quantities correlated with the CCIF (infectious virus titers) results (R² = 0.98).

DT40 and Jurkat cells were settled onto poly‐l‐lysine (Sigma‐Aldrich)‐coated glass coverslips, whereas HEK 293T, 293 Flp‐In T‐REx and RPE‐1 cell lines were grown on uncoated coverslips. Cells were fixed with 100% −20°C methanol or with 4% paraformaldehyde (Polysciences) in phosphate‐buffered saline (PBS) for 10 min followed by 5 min in 100% 20°C methanol (ACROS Organics). Cells were permeabilised in PBS‐0.5% Tween‐20 (Promega; PBS‐T) or, for centriolar staining, in 0.5% Triton X‐100 (ACROS Organics), 0.05% sodium dodecyl sulphate (SDS; Sigma‐Aldrich) and 0.5% Tween‐20 (Promega) in PBS for 5 min. Cells were then stained as described in Sir et al (2013). Primary antibodies are listed in the reagents and tools table in Appendix Supplementary Methods. Secondary antibodies conjugated to Alexa Fluor 488, 555 or 647 were obtained from Invitrogen. To visualise DNA, coverslips were incubated with 1 μg/ml Hoechst 33258 (Sigma‐Aldrich) and then mounted on glass slides (SuperFrost Ultra Plus, Thermo Scientific) using the ProLong Diamond Antifade Mountant (Invitrogen).

The DNA fragments used in this study, listed in Supplementary Table S1, were chemically synthesized with an H8 DNA/RNA Synthesizer (K&A Laborgeraete), by using natural-base and biotin-dT phosphoramidites from Glen Research and a Ds phosphoramidite synthesized in-house, as described previously (42 (link)). The chemically synthesized DNAs were purified by denaturing gel electrophoresis. The mouse monoclonal anti-VEGF antibodies (clones 26503, VG76e, 16F1 and VG1) and anti-IFNγ antibodies (clones B133.5 and 2G1) were obtained from Thermo Fisher Scientific. Rabbit anti-biotin IgG (A150-109A) was purchased from Bethyl Laboratories, Inc. Recombinant human VEGF₁₆₅ and IFNγ were obtained from PeproTech. The streptavidin-HRP conjugate (1 mg/ml) and the anti-mouse IgG HRP conjugate (1 mg/ml) were obtained from Jackson ImmunoResearch and Promega, respectively. Stock solutions (10×) for PBS and D-PBS(–) were obtained from Thermo Fisher Scientific and Nacalai Tesque, respectively. BSA, streptavidin, and Tween 20 were purchased from Promega. Nonidet P-40 was purchased from Nacalai Tesque. The TMB-substrate solution was purchased from KPL. Human serum (lot # SLBS8635) was obtained from Sigma-Aldrich.

The water used in the preparation of all solutions was ultra-pure. When not disclosed, reagents are from Fischer Scientific. A TRIS-EDTA (TE) buffer was prepared by combining TRIS, ethylenediaminetetraacetic acid (EDTA), and K₂HPO₄ at 10 mM, 1 mM, and 100 mM concentration, respectively. pH was adjusted to 7.4 using 1 M HCl. A TRIS-borate-EDTA (TBE) buffer 10x was acquired from FRILABO (Porto, Portugal). Phosphate buffer (PB) 0.1 M, pH 7.2 was prepared from stock solutions of Na₂HPO₄ and NaH₂PO₄ at 0.2 M. PB-Tween20 consisted on PB buffer with 0.02% (v/v) of Tween^® 20 from Promega (Madison, WI, USA).
The customized single stranded oligonucleotide sequences were synthesized by STABVIDA (Caparica, Portugal).
DNA-free water, MasterMix 16S Basic, and Moltaq 16S from Molzym (Germany) were used for PCR reactions. Electrophoresis reagents included TopVision agarose from Thermo Fisher Scientific^TM (MA, USA), for gel preparation, GRS DNA loading buffer blue 6x from GRiSP (Porto, Portugal), GeneRuler 1 kb DNA ladder from Thermo Fisher Scientific^TM, and Diamond^TM nucleic acid dye from Promega.
The MNPs were nanomag^®-D from Micromod (Rostock, Germany), with a diameter of 250 nm and 75–80% (w/w) magnetite in a matrix of dextran (40 kDa), streptavidin coated. The particles have a magnetic moment of ∼1.6 × 10⁻¹⁶ Am² for a 1.2 kA/m magnetizing field and a susceptibility of χ ∼ 4.