Cd80 pe clone l307.4

The levels of MHC class II and costimulatory molecules expressed on monocytes and pDCs were assessed by flow cytometry from their overnight culture with DNA-LL37 complexes or DNA alone, pDCs were surface stained with lineage negative cocktail (FITC labelled anti-human CD3, CD14, CD16, CD19, CD20, CD56), anti-human BDCA-2 FITC or BDCA-2 PE (clone AC144) (Miltenyi biotec GmBh), CD123 PE (clone 7G3), HLA-DR, DP, DQ BV421 (clone Tu39), Immunoglobulin-like transcript 7 (ILT7) allophycocyanin (APC) (Clone 17G10.2), CD80 PE (clone L307.4) and CD86 PE Cy7(clone 2331 FUN-1) antibodies (BD Biosciences). Similarly, monocytes were stained by anti-human CD14 Peridinin Chlorophyll Protein (PerCP) (clone MΦP9) and HLA-DR, DP, DQ BV421, CD80 and CD86 antibodies (BD Biosciences).

For FACS nonspecific binding was blocked using human TruStain FcX (BioLegend). Macrophages were stained with Zombie aqua, CD3-APC-Cy7 clone HIT3a CD86-APC and PE-Cy5 clone IT2.2, HLA-DR-FITC and APC clone L243 (BioLegend), CD14-BV650 and BUV737 clone M5E2, and CD80-PE clone L307.4 (BD Pharmingen^TM) and then acquired by flow cytometry. The FACS data were analyzed with FlowJo software (TreeStar Inc.).

At 24 h post transfection, macrophages were harvested by scraping, then washed with cold autoMACS running buffer (Miltenyi Biotec). Cells were incubated with FcR blocking reagent (Miltenyi Biotec) for 10 min at 4°C, to avoid unspecific antibody binding. Upon washing, cells were stained with CD80-PE (clone L307.4) (BD Bioscience, Heidelberg, Germany) antibody with dilution factor 1:100 (5 μg/mL final concentration) at 4°C for 10 min. After a final washing step, cells were analyzed with MACSQuant VYB (Miltenyi Biotec). For live-dead discrimination, DAPI, at a final concentration of 1 μg/mL, was added to each sample immediately before measurement. All flow cytometric data were analyzed by FlowJo software V10 using the previously established gating strategy.⁴¹ (link) Briefly, cells were initially identified from debris by gating on forward versus side scatter area (FSC-A versus SSC-A) dot plots, followed by exclusion of aggregated cells using forward scatter area against height (FSC-A versus FSC-H). DAPI-negative cells were identified as live cells. EGFP-positive cell populations were determined among live single cells, by gating with respect to untransfected negative controls.