Cell strainer

For blood withdrawal, 72 non-irradiated zebrafish were anesthetized using 0.04% MS-222 in system water. Thereafter, a big portion of their tail was cut using a straight razorblade. The emerging blood was collected with a pipette and stored in 100 μl PBS in heparin-coated tubes (Microvette, Sarstedt, DE). For the frequency-assessment of the fli1a⁺ fraction, the blood was filtered with 40 μl cell strainers (Sigma-Aldrich, CH) and data directly acquired using FACSVerse (BD Biosciences). For the labeling of different surface cluster of differentiations (CD molecules), the cell isolates were fixed in 2% paraformaldehyde for 15 min, washed in PBS with 2% BSA (Sigma-Aldrich) and incubated with primary antibodies for 20 min at 4 °C. The secondary antibody was applied for 1 h at 4 °C. Stained cells were acquired using FACSVerse (BD Biosciences). The rabbit anti-zebrafish monoclonal antibodies against CD4 and CD8 were purchased from Anaspec, USA. The secondary donkey anti-rabbit AlexaFluor647-labeled antibody was purchased from Abcam, CH. FloJo Version 9.8.1 was used for data analysis. For blood smears, the blood was spread on a glass slide, air-dried for 3 min and then observed with a Leica M205FA stereomicroscope to identify fli1a⁺ cells. Thereafter, the cells were immediately stained with Giemsa-May Grünwald (Grogg Chemie, CH).

Two nasal swabs (one per nostril) were taken daily following infection with H1N1pdm09. Tonsil, mesenteric, tracheobronchial (TBLN) and mandibular lymph nodes were dissected out, cut into smaller pieces and tissue integrity disrupted by squashing them with the back of a plunger in the presence of RPMI supplemented with 10% FBS. The tissue homogenates were passed through 100 μM cell strainers (Sigma, UK). Blood, spleen, broncho-alveolar lavage (BAL) and accessory lung lobe were processed as described previously [33 (link),41 (link)]. Virus titer in nasal swabs, BAL and lung was determined by plaque assay on MDCK cells as previously described [33 (link)].

Primary NSCs were isolated from the whole brain excluding cerebellum of postnatal day 0 (P0) mice or from the subgranular zone (SGZ) of 2-month-old adult mice as described previously (Zhu et al., 2019 (link)). Briefly, the forebrains from P0 mice or the dentate gyrus from adult mice were dissociated with papain (Worthington) and DNase I (Sigma) and then undissociated cell clusters were removed by a cell strainer (Sigma). Dissociated cells were cultured in serum-free DMEM-F12 (Gibco) supplemented with B27 supplement (Gibco), 2 mM L-glutamine (Gibco), 20 ng/ml EGF (PeproTech), and 20 ng/ml FGF2 (PeproTech). After glial cells and neurons died, primary NSCs were maintained as neurospheres in uncoated dishes.
C17.2 mouse NSC line was purchased from Sigma and cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 2 mM L-glutamine (Gibco).

Tissue samples were collected at Day 77 (7 days post second rMVA booster, rectal biopsies) and at autopsy (rectal and cerivo-vaginal tissues). All mucosal tissues samples were stored in RPMI-1640 (Sigma) supplemented with 25% foetal bovine serum until use the same day. Single cell suspensions were prepared for intracellular cytokine staining (ICS) as described previously^{39 (link),43 (link),47 (link)}. Briefly, tissue samples were mechanically cut into small pieces and incubated in complete RPMI (10% FBS) containing 2 mg/mL collagenase (Sigma), 2.4 mg/mL dispase (Gibco-Invitrogen) and 5 Units/mL DNAse (Calbiochem La Jolla, CA), for 45 min at 37 °C, with regular vortexing every 10 min, digested tissue was passed through two layers of sterile gauze to remove debris, centrifuged and resuspended in erythrocyte lysis buffer (0.15 mM NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA) for 2 min at room temperature. Cells were washed twice in complete RPMI and passed through a 100 μm cell strainer (Sigma) to remove any remaining debris. Finally, cells were centrifuged and resuspended in 0.5–1 mL of complete RPMI.

Femurs, tibias and ilium were dissected and crushed with a scissors and a pestle. The crushed bones were gently washed once in HBSS+ (Hanks-balanced salt solution supplemented with 2% FBS, 10 mM Hepes, and 100 U/mL penicillin/ 0.1 mg/mL streptomycin solution), and the solution filtered through a cell strainer (BD Falcon) was discarded. The bone fragments were collected and incubated for 1 h at 37 °C in 20 mL of DMEM (Invitrogen) containing 0.2% collagenase (Wako Chemicals USA, Inc.), 10 mM Hepes and 100 U/mL penicillin/ 0.1 mg/mL streptomycin solution. The suspension was filtered with a cell strainer (BD Falcon) to remove debris and bone fragments, and collected by centrifugation at 400 g for 5 min at 4 °C. The pellet was immersed in 1 mL water (Sigma-Aldrich) for 5–10 s to burst the red blood cells, after which 1 mL of 2 × PBS (diluted the product from Sigma-Aldrich) containing 4% FBS was added, and the suspension was filtered through a cell strainer. These serial procedures are described in previous report^{28 (link)}.

During delivery, splenic tissues were maintained at 15–22°C and RPMI 1640 (Thermo Fisher Scientific) supplemented with penicillin-streptomycin (100 U/mL, Thermo Fisher Scientific) was used as delivery medium. Spleens were comminuted mechanically in a culture dish containing RPMI with penicillin-streptomycin (100 U/mL, Thermo Fisher Scientific), filtered through a cell strainer (70 μm, Sigma Aldrich). After decantation, the cell suspension was transferred into a 50 mL tube (Falcon) containing Pancoll (Pan Biotech) and centrifuged. Then, cells were washed twice with RPMI medium and frozen at −150°C in medium containing fetal bovine serum (FBS) (Sigma Aldrich) with 10% dimethyl sulfoxide (DMSO) (Sigma Aldrich).