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.
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.
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