To measure binding capacity, beads were stained with biotinylated PE at saturating concentration (50 nM) in PBST, and their fluorescence measured by flow cytometry (FACSCalibur, BD Biosciences).
After washing with Annexin V binding buffer, cells were stained with 2000-fold diluted Cy5-labeled AnnexinV (Biovision, Milpitas, CA) and 500 nM Sytox blue (Invitrogen) and analyzed by flow cytometry.
For visualization and spot isolation of plasminogen-binding proteins, blots were stained with India Ink.
Then, binding buffer was replaced and cells were stained with PI.
They were resuspended at 10 cells/ml in binding buffer, 100 μl of cells were stained with 5 μl Annexin-V and 5 μl propidium iodide, and incubated in the dark for 15 min at room temperature, as recommended by the manufacturer.
To control for possible nonspecific binding of the antibodies, additional samples were stained with either a directly conjugated antibody of the same isotype as the primary antibody, or unconjugated antibody of the same isotype followed by streptavidin biotin or streptavidin biotin alone.
The nuclei were stained with DNA-binding dyes Hoechst no.
To determine whether tetramer+CD8low/− cells were possibly NK cells non-specifically binding MHC class I tetramers, tissue sections were stained with negative control tetramers loaded with a peptide derived from the hepatitis B virus core.
For the binding competition assay 1×106 or 5×105 DCs were stained with the above anti-CD11b and anti-CD11c antibodies and incubated overnight on ice with different amounts of unlabeled CPMV (0 µg, 1 µg, 10 µg, 100 µg, and 1000 µg).
