Ls positive selection column

H1N1/WSN virus (A/WSN/1933(V-1520)) was purchased from American Type Culture Collection (ATCC). The LPAI isolates H5N2/F118 (A/Duck/Malaysia/F118/2004), H5N3 (A/Duck/Singapore-Q/F119/1997) and H9N2 (A/Duck/Malaysia/02/2001) viruses were obtained from Agri-Food and Veterinary authority of Singapore and have been characterized previously [31 (link)]. The 9 to 11-day old embryonated chicken eggs were used for virus stock preparation, and TCID50 in MDCK (standard plaque assay) was performed to measure the infectivity of the viruses. PMФ (CD11b+) cells were obtained from 6-8 pathogen-free female BALB/c mice lungs as described before [32 (link)]. Briefly, single cell suspensions (0.5% BSA, 2 mM EDTA, in 1xPBS) obtained from digested murine lungs (with collagenase D (1 mg/ml; Gibco) was passed through a 30 μm filter. CD11b microbeads and a LS positive selection column (MiltenyiBiotec) were used to purify CD11b + cells. The purified cells were confirmed by Trypan blue viability assay and more than 95% of them were viable. L929 cell (conditioned 30%v/v) medium was used to maintain the purified cells at 37 °C in 5% CO₂. The cells were washed with PBS to remove non-adherent cells before used for virus infection. The NP, F4/80-FITC and CD11b antibodies were purchased from Chemicon, Biolegend and MiltenyiBiotec respectively. Cells were infected with each virus using a MOI of 5.

Cells were dissociated from the monolayer using TrypLE Express (Gibco) and washed 1X in PBS/10% fetal bovine serum (FBS; Wisent Inc.) (FACS buffer) containing 10 μM Y27632 (Tocris). Live cells were incubated for 20 min at room temperature with an anti-GP2 antibody in FACS buffer. After washing once with FACS buffer, the live cells were incubated for an additional 20 min at room temperature with an anti-mouse PE-conjugated secondary antibody in FACS buffer. The cells were washed once again with FACS buffer, and re-suspended in MACS buffer (Miltenyi Biotec) with anti-PE microbeads (Miltenyi Biotec) at a concentration of 75 μl MACS buffer and 20 μl beads/1 × 10⁷ cells. They were incubated at 4 °C for 15 min, followed by one wash in MACS buffer. The cells were then loaded onto a LS positive selection column (Miltenyi Biotec) and the flow through collected. The column was washed thrice and the cells eluted, all in FACS buffer with 10 μM Y27632 (Tocris). The elution from the LS positive selection column formed the GP2⁺ fraction. The flow through from the LS positive selection column was then loaded onto a LD depletion column (Miltenyi Biotec). The flow through from the LD depletion column was used as the flow through fraction. Following MACS, the cells were cultured as aggregates. Supplementary Table 3 for list of antibodies used.

Lympholyte^®‐Mammal cell separation reagent was from CedarLane, Burlington, ON, Canada. Anti‐PE MicroBeads, LS+ Positive Selection Columns and QuadroMACS™ Separator were from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany. CountBright™ Absolute Counting Beads were from Invitrogen, Life Technologies™, Carlsbad, CA. Heat‐inactivated foetal bovine serum (FBS) was from PAA Laboratories, Pasching, Austria. Anticoagulant Solution, Buffered Salt Solution, Nucleic Acid Dye Solution (SYTO^®13), Anti‐CD24‐PE and Anti‐CD61‐PE were from the MutaFlow^PLUS kit (mouse blood, tube‐based). This kit and the micronucleus analysis kit (In Vivo Mouse MicroFlow^PLUS kit) were from Litron Laboratories, Rochester, NY. N‐Nitroso‐N‐Ethylurea (ENU, cat. no. 3385) was purchased from Sigma–Aldrich Norway AS, Oslo, Norway. Low melting point agarose (NuSieve^®GTG^®Agarose) and Gelbond^® films were from Lonza, Rockland, ME. SYBR^®Gold Nucleic Acid Gel Stain (10,000× concentrate in DMSO) was from Life Technologies™, Carlsbad, CA.