Streptavidin alexa 488

The analysis of DCs on flow cytometer (Coulter, XL-MCL, Krefeld Germany) or confocal microscope (Zeiss LSM 510/Axiovert 200 M, Jena, Germany) was performed upon labeling the cells with primary antibodies (Abs). The following Abs (clones) and reagents were used for the immunocytochemistry and flow cytometry: IgG1a negative control–biotin (MCA928), anti-CD1a-phycoerythrin (PE) (NA1/34HLK), IgG1 negative control–PE (MCA928PE), anti-CD14-Fluorescein isothiocyanate (FITC) (TUK4), anti-CD86–FITC (BU63), anti-CD3-FITC (UCHT1), IgG1 negative control–FITC (MCA928F) (all from Serotec, Oxford, UK), anti-CD45-PECy5 (HI30), anti-HLA-DR-biotin (LN3), IgG1a negative control-PECy5 (P.3.6.2.8.1) (all from eBioscience), streptavidin-Alexa 488, anti-CD83-Alexa 488 (all from Biolegend). The expression of HLA-DR, CD86 and CD83 was analyzed within CD45⁺ DC population. Necrosis of DCs cultivated with GNPs (5–200 µg/ml) was measured after 48 h-cultures by staining the cells with propidium iodide (PI, 10 µg/ml Sigma) in phosphate buffer saline (PBS). Apoptosis was determined after 48 h by staining the cells with PI in hypotonic citric/Triton-X buffer, or after 24 h by Annexin-V-FITC/PI (R&D) labeling.

For immunofluorescence experiments, tumors were harvested, washed, and fixed in Antigenfix solution (Diapath) for 2 h with agitation. After several steps of PBS 1X washing, samples were placed in PBS containing 30% sucrose overnight and included in OCT. After saturation with PBS 2% BSA, samples were stained by overnight incubation at 4°C with anti-CD8b (Clone 53–5.8; BioXcell) and MHC-II (2G9; BD-Pharmingen) mAbs. After incubation with donkey anti-rabbit Cy3 serum and streptavidin Alexa488 (BioLegend), samples were mounted in a mounting medium. Images were acquired using a Zeiss LSM780 confocal microscope and analyzed using ZEN 2.3 software.