Anti cd80 fitc

Flow cytometry was performed as previously described [26 (link),27 (link)]. Analysis of the expression levels of cell-surface markers was performed by flow cytometry. DCs were blocked for 5 min with PBS containing 10% heat inactivated human AB serum (Sigma-Aldrich, St. Louis, MO, USA), and then stained by adding the antibodies to the same buffer. All other cell types were stained on PBS containing 3% FBS. Cells were labeled with anti-CD1a-PE, anti-CD3-PC7, anti-CD4-FITC, anti-CD14-PC7, anti-CD19-ECD, anti-CD25-PC7, anti-CD69-PC5, anti-CD80-FITC, anti-CD86-PC5.5, anti-CD86-PE, anti-HLA-DR-ECD, anti-HLA-DR-PB, anti-HLA-DR-PE (all from Beckman Coulter Inc., Brea, CA, USA), anti-CD8-Pacific Blue, anti-CD71-APC-Cy7, anti-CD197-APC-Cy7 (CCR7) (all from BioLegend, San Diego, CA, USA), anti-CD83-APC (BD Biosciences, Franklin Lake, NJ, USA), and anti-CD3-PO (Abcore, Ramona, CA, USA).
Levels of surface expression on cells were estimated by flow cytometry (Gallios; Beckman Coulter, Brea, CA, USA) and analyzed using Kaluza software (Beckman Coulter, Brea, CA, USA). Entry of FITC-labeled p24 peptide into the DCs was also estimated by flow cytometry. DCs were collected 2 h, 24 h, and 48 h after complex addition, washed with PBS, and acid washed, in order to eliminate any peptide attached to the cell surface. Fluorescence was measured by flow cytometry.

Analysis of cell-surface phenotype of DCs and MØs was performed by flow cytometry. Cells were labeled with anti-CD4-FITC, anti-CD69-PC5, anti-CD80-FITC, anti-CD1a-PE, anti-HLA-DR-ECD, anti-CD14-PC7, anti-CD86-PC5.5 (all from Beckman Coulter Inc., Brea, CA, USA), anti-CD8-Pacific Blue, anti-CD36-APC, anti-CD197-APC-Cy7 (CCR7) (all from BioLegend, San Diego, CA, USA), anti-CCR5-PE, anti-CD209-PE, anti-CD83-APC (all from BD Biosciences, Franklin Lake, NJ, USA), anti-CXCR4-APC (RandD systems, Minneapolis, MN, USA) and anti-CD163-FITC (MBL International Corp., Woburn, WA, USA). Levels of surface expression on cells were estimated by flow cytometry (Gallios; Beckman) and analyzed using Kaluza software (Beckman).