Rosettesep monocyte enrichment kit

Monocytes were isolated from buffy coats using Ficoll centrifugation (Lymphoprep; Axis Shield) after negative selection using the RosetteSep Monocyte Enrichment Kit (StemCell Technologies). Monocyte-derived DC (MoDC) were obtained after 6 days of differentiation in complete RPMI medium (RPMI 1,640, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM L-glutamine, 1% Penicillin/Streptomycin, Hyclone GE Healthcare, and 10% FBS, Sigma) complemented with GM-CSF (250 ng/mL; PeproTech) and rIL-4 (6.5 ng/mL; R&D Systems). Cells were seeded at a density of 5 × 10⁵ cells/mL and after 3 days of differentiation, 50% of the medium was replaced and new cytokines added. Staining for flow cytometry was done before and at indicated time points post viral infection. MoDC were incubated with LIVE/DEAD® Fixable near-IR Dead Cell Stain Kit (Life Technologies) followed by staining with CD14-PE-Cy7 (MφP9), CD1a-BV510 (HI149), CD80-PE (L307.4), and CD86-APC (2331 FUN-1) from BD Biosciences. The mouse anti-IAV NP mAb (H16-L10-4R5; Merck Millipore) was detected with a secondary Ab coupled to Alexa Fluor 488 fluorochrome with the Zenon® Kit (Invitrogen). Acquisition was done on a Fortessa flow cytometer (BD Biosciences) and analysis was performed with FlowJo software (Tree Star, version 10.2).

Human Buffy coats were acquired from Karolinska Institutet, Stockholm, Sweden. All experimental work with human peripheral blood cells were carried out in accordance with Swedish guidelines and regulations. All experimental protocols were approved by the local ethical committee in Stockholm (“Regionala etikprövningsnämnden i Stockholm”, Ethical permit Dnr 2006/229-31/3). According to regulations in Sweden, experimental in vitro work with cells from buffy coats does not require informed consent. Human monocytes were generated using the RosetteSep Monocyte Enrichment Kit (1 mL/10 mL buffy coat; StemCell Technologies) and differentiated into moDC, with GM-CSF (250 ng/mL; PeproTech) and IL-4 (6.5 ng/mL; R&D Systems) for 6 days in +37 °C, 5% CO₂ at a density of 5 × 10⁵ cells/mL in RPMI 1640 completed with 10% FCS, 1 mM sodium pyruvate, 10 mM HEPES, 2mM L-glutamine, and 1% streptomycin and penicillin (all from Invitrogen Life Technologies) as previously described^{17 (link)}. Differentiation of the cells was monitored by CD1a expression. PBMCs were isolated from buffy coats after Ficoll separation (Stemcell).

Human monocytes were negatively selected from buffy coats using the RosetteSep Monocyte Enrichment Kit (1 mL/10 mL buffy coat; StemCell Technologies) and differentiated into moDC, using granulocyte-machrophage colony-stimulating factor (250 ng/mL; PeproTech) and interleukin-4 (6.5 ng/mL; R&D Systems) for 6 days in 37 °C, 5% CO₂ at a density of 5 × 10⁵ cells/mL in RPMI 1640 completed with 10% FCS, 1 mM sodium pyruvate, 10 mM HEPES, 2 mM l-glutamine, and 1% penicillin/streptomycin (ThermoFisher, USA). Immature moDC were exposed to RSV pre-incubated with different biological fluids for 4 h in serum-free media, washed, and then incubated in serum-containing medium for 72 h before analyses of CD86 and GFP expression. Dead cells were excluded using Live/Dead fixable near-IR dead cell stain kit (ThermoFisher). Flow cytometry sample data were acquired on a FACSVerse (BD Biosciences) and the analysis was performed in FlowJo software (TreeStar).