Recombinant mouse ifn γ

For this test, the pHrodo Red E. coli BioParticles Conjugate for Phagocytosis assay was used (Thermo Fisher Scientific, #P35361). The macrophages were pre-activated with 20 ng/mL recombinant mouse IFN-γ (BD #554587) for 48 h and then received the following treatments: PBS only (M0 profile control), E. coli O26:B6 LPS 1 µg/mL (used as a canonical positive control of macrophages activation to M1 profile^{21 (link)} to compare with PnV effects), or PnV 14 µg/mL. After 24 h, treatments were removed, macrophages were washed with PBS and incubated for 4 h with 50 µg bioparticles. The cells were then extensively washed, fixed with paraformaldehyde for 15 min and labeled with Iba-1 by immunofluorescence. Briefly, cells were incubated with blocking buffer (PBS with 1% BSA and 0.3% Triton X100) for 2 h, followed by anti-Iba-1 (Wako #019-19741; 1:700) overnight, and secondary antibody (Cy2-Goat anti-rabbit, Jackson ImmunoResearch, #111225144; 1:1000) for 1 h. Nuclei were stained with DAPI (1:1000) for 5 min. Macrophages were washed and sliders were mounted using a mounting medium for fluorescence. Images were acquired in the Cytation 5 – Cell Imaging Multi-Mode reader (BioTek, Winooski, VT, USA). For quantification, images of three randomly selected fields per treatment were taken and integrate analysis of pixels was determined using Image J software (version 1.52p, NIH, USA).

JAWSII (ATCC^® CRL-11904™) murine dendritic cells and fetal bovine serum (FBS) were purchased from ATCC (Manassas, VA, USA). Because it is an immortalized, commercially available cell line, the use of JAWSII dendritic cells was exempted from approval by Virginia Tech Institutional Animal Care and Use Committee. Gibco™ alpha minimum essential medium (MEM α) containing ribonucleosides, deoxyribonucleosides, and L-glutamine, Gibco™ recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF), Gibco™ 0.25% Trypsin-EDTA solution, Invitrogen™ TRIzol™ Reagent, Invitrogen™ MultiScribe™ Reverse Transcriptase, 1 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) solution, and bovine serum albumin (BSA) were purchased from ThermoFisher Scientific (Waltham, MA, USA). Phycoerythrin (PE) linked anti-mouse CD86 antibody and flow staining buffer were purchased from BioLegend (San Diego, CA, USA). Poly I:C sodium salt and gelatin from porcine skin were purchased from Sigma–Aldrich (St. Louis, MO, USA). R848 was purchased from InvivoGen (San Diego, CA, USA). Recombinant mouse IFN-γ was purchased from BD Biosciences (Franklin Lakes, NJ, USA).