Cd54 apc

Antibodies CD11b V450, CD16 PE-Cy7, CD18 PE, CD32PerCP-efluor 710, CD54 APC and CD64 APC-H7 were purchased from BD Bioscience (Heidelberg, Germany) and eBioscience (Frankfurt, Germany). As lineage markers, CD66b-FITC (granulocytes) from Beckman Coulter (Krefeld, Germany), CD3-eFluor 450 (T cells) from eBioscience, CD14-APC-Cy7 (monocytes) from BD Bioscience and CD56-BV510 (NK cells) from BioLegend (Fell, Germany) were used. Appropriate isotype antibodies from eBioscience, BD Bioscience and BioLegend were used as controls (see also S1 Table). Cells were incubated with antibodies for 30 min at 4°C and acquired on a BD FACS Canto II flow cytometer. Analysis and calculations were performed with BD FACS DIVA software.

For mCD40L expression in CFPAC-1 cells transduced with RAdnCD40L, 3 × 10⁵ cells were washed three times with ice-cold PBS buffer and incubated on ice for 20 min with 100 μl of diluted mouse anti-human CD40L-APC conjugate Ab or mouse isotype-APC Ab conjugate (ebioscience, San Diego, CA, USA) or left without treatment as negative control. Cells were then washed three times with ice-cold staining buffer (PBS with 1%BSA and 0.1% NaN3) analysed by flow cytometry. For monocyte-derived DC activation markers, mouse anti-human mAb CD1a- PE-Cy7, CD14-PE, CD40-APC, and HLA-DR-Alexa Fluor 700 were from ebioscience. For mouse anti-human mAb CD86-PE, CD83-APC and CD54-APC, were from BD bioscience, San Jose, CA, USA. Briefly, DC were stained with mouse anti-human mAbs or the isotype control for 20 minutes, cells were washed twice with staining buffer. Flow cytometry was performed by acquiring cells using BD LSR Fortessa cell analyser (BD Bioscience). A minimum of 50,000 HLA-DR + cells were acquired per sample and data were analysed by FlowJo software version X (Tree Star, Ashland, USA)