Macs cd11c labeled magnetic beads

GM-BM were generated from murine bone marrow precursors using recombinant mouse (rm) GM-CSF. Cells were obtained as previously described [65 (link)] with minor modifications. GM-BM were cultured in RPMI-1640 (HyClone, Logan, UT, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone), 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), 50 μM 2-mercaptoethanol (Sigma-Aldrich) and 20 ng/ml rmGM-CSF (R&D Systems, Minneapolis, MN, USA). After 10 days of culture in 100 mm bacteriological Petri dishes (Corning [Falcon] BD, Bedford, MA, USA) GM-BM were separated using MACS CD11c⁺ labeled magnetic beads (Miltenyi Biotec, Auburn, CA, USA) [26 (link)]. After MACS separation (purity ≥ 95%) CD11c⁺ cells were exposed to: (a) complete RPMI-1640 medium (mock, negative control), (b) live-ECTV [multiplicity of infection, m.o.i. = 5], or (c) uvi-ECTV (m.o.i. = 5, before inactivation). After 60 min virus adsorption at 37°C in a humidified 5% CO₂ atmosphere, mock-, ECTV- or uvi-ECTV-exposed cells were cultured in the absence or the presence of LPS (Escherichia coli 0111:B4; Sigma-Aldrich, St Louis, MO, USA) for 24 h at a final concentration of 1μg/ml, which served as a control of GM-BM maturation.

The protocol used to generate GM-BM was similar to that used by Lutz et al. (1999) (link). Bone marrow was flushed with cold RPMI-1640 medium from femurs and tibias of mice and erythrocytes were removed using ammonium chloride buffer. Cells were then washed and plated at 1 × 10⁶/well in a six-well plate in RPMI-1640 medium containing 10% heat-inactivated FBS, 1% antibiotic solution (100 U/ml penicillin and 100 mg/ml streptomycin; Sigma–Aldrich), 50 μM 2-mercaptoethanol (Sigma–Aldrich), and 20 ng/ml recombinant mouse (rm) GM-CSF (R&D Systems, Minneapolis, MN, United States). Fresh medium with 20 ng/ml rmGM-CSF was added at day 3 of culture and then was partially replaced every 2 days. On day 8 post-culture, cDCs were enriched using MACS CD11c⁺ labeled magnetic beads (Miltenyi Biotec, Auburn, CA, United States). After MACS separation GM-BM cultures were evaluated for surface expression of cellular markers using the following monoclonal antibodies (mAbs): anti-mouse CD11c-BV421 (N418, Armenian Hamster IgG2; BioLegend, San Diego, CA, United States), anti-CD11b-BV605 (M1/70, rat IgG2b), anti-I-A/I-E-BV711 (M5/114.15.2, rat IgG2b; both from BD Biosciences, San Jose, CA, United States) and anti-CD205-PE-Cy7 (NLDC-145, rat IgG2a; BioLegend) (Supplementary Figure S1).

Primary culture of BMDCs was obtained using mouse recombinant granulocyte-macrophage colony stimulating factor (rmGM-CSF; Sigma-Aldrich, St. Louis, MO, USA), as described previously [45 (link)] with minor modifications. Bone marrow isolated from the tibia and femur was suspended in RPMI 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 1% antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin, Symbios, Gdansk, Poland). Erythrocytes were lysed with NH₄Cl-Tris buffer. Cells were resuspended in R-10 growth medium [RPMI-1640 medium enriched with 10% heat-inactivated fetal bovine serum (FBS, HyClone, Logan, UT, USA), 1% antibiotic solution (100 U/mL penicillin and 100 μg/mL streptomycin, Symbios), 50 μM 2-mercaptoethanol (Sigma-Aldrich), and 20 ng/mL rmGM-CSF], and then placed into wells of a six-well plate and incubated at 37 °C in the presence of 5% CO₂. The growth medium was added sequentially on day 3 of culture and replaced partially on day 6 of culture. On day 8 of culture, BMDCs were enriched using MACS CD11c⁺-labeled magnetic beads (Miltenyi Biotec, Auburn, CA, USA). Cell viability was above 95%, as determined by the 0.4% trypan blue exclusion test.