Cd274 apc

For detection of branched complex glycoconjugates, cells were stimulated with 50 ng/mL LPS for 24 h and 48 h hours or left untreated. Cells were washed in ice-cold 1% bovine serum/PBS and incubated with 2 µg/200,000 cells Alexa488-coupled PHA-L lectin (Invitrogen). For the detection of differentially expressed glycoproteins after LPS treatment, cells were stimulated for 4 h, 24 h, or 48 h with or without LPS and labelled with specific fluorescent-dye conjugated antibodies CD54-APC, Immunotools; CD14-FITC, CD169-APC, CD86-PerCP-Cy5 CD127-APC, CD274-APC, CLEC12A-PE, all eBiosciences; CD319-Alexa647, BD Biosciences; CD80-PE, Miltenyi Biotec). After washing, flow cytometry was performed with an Attune Acoustic Focusing Cytometer Attune (Applied Biosystems) or FACS-ARIA II (BD Biosciences). For visualization, FCS-files were loaded into FlowJo. Expression of glycoprotein marker proteins was analyzed by gating on live CD14⁺ cells.

The following directly conjugated anti-human monoclonal antibodies were used: CD4-FITC, CD4-APC, CD8-PE, CD163-PE, CD206-FITC, CD86-PE-Cy5, CD64-APC, CD273-PE, TLR-4-PE-Cy7, CD40-FITC, HLA-DR-PECy5 and CD274-APC (eBioscience, San Diego). Saturating amounts of antibody were used to stain approximately 3 x 10⁵ cells in staining buffer (1 x PBS, 2% FBS) at a final volume of 20 μl for 30 min at 4°C protected from the light. All samples were washed with staining buffer and resuspended in 200 μl of FACS Buffer. Samples were examined in a FACSCalibur (BD Biosciences) and analysis was performed using FlowJo software (Tree Star, Inc., OR).