Anti cd14 pecy5

PBMC were collected from the co-culture by careful pipetting. Monocyte
activation was assessed by staining with ethidium monoazide (EMA), anti-CD19-PeCy5,
anti-CD14-PB, anti-CD3-AlexaFluor700 and anti-CD16-Violet500, anti-HLA-DR-FITC and
anti-CD69-PE (all from BD Biosciences). NK cell function was assessed after incubation
with brefeldin A for the final 3h of the 24h co-culture with Huh7/HCV-replicon cells or
Huh7 cells. Alternatively, lymphocytes were removed and exposed to IL-12 (0.5 ng/ml) and
IL-15 (20 ng/ml; both from R&D Systems, Minneapolis, MN) for an additional 18h
with addition of brefeldin A for the last 4h as described ^{2 (link)}. Cells were then washed and stained with EMA,
anti-CD19-PeCy5 (BD Biosciences), anti-CD14-PeCy5 (Abd-Serotec, Raleigh, NC) to exclude
dead cells, B cells and monocytes, and with anti-CD3-AlexaFluor700, anti-CD56-PeCy7 and
anti-CD16-PacificBlue to identify NK cells. Cells were washed again, fixed and
permeabilized with the Cytofix/Cytoperm Kit, stained with anti-IFN-γ-Pe (BD
Biosciences), washed, and immediately analyzed on an LSRII flow cytometer using FacsDiva
Version 6.1.3 (BD Biosciences) and FlowJo Version 8.8.2 (Tree Star, Ashland, OR)
software.

Thawed PBMC were stained with ethidium monoazide (EMA), anti-CD19-PeCy5, anti-CD3-AlexaFluor700 (both from BD Biosciences), and with either anti-CD14-V711 (Biolegend) or anti-CD14-PeCy5 (AbD Serotec, Raleigh, NC) to exclude dead cells, T cells, B cells and monocytes. NK cells were identified using anti-CD56-PeCy7 (BD Biosciences). FITC-conjugated antibodies against CD122, CXCR3 (R&D Systems), CD69 and HLA-DR (BD Biosciences), PE-conjugated antibodies against TRAIL, CD300 (BD Biosciences), NKG2A, NKG2D, and NKp44 (Beckman Coulter, Brea, CA), and APC-conjugated antibodies against CD85j (eBiosciences, San Diego, CA), CCR5 (BD Biosciences), NKG2C (R&D Systems), NKp30 and NKp46 (Miltenyi Biotec, Auburn, CA) were added. Liver-infiltrating lymphocytes were stained with anti-TRAIL-PE, anti-CD69-APC/Cy7 (BD Biosciences) and anti-NKp46-APC (Miltenyi Biotec) in addition to EMA and the lineage-specific antibodies described above.