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After secondary antibody binding the cells were washed three times with ice-cold PBS-G and then resuspended in 2 ml of ice-cold PBS-G, passed again through a 40 micrometer filter and then immediately analyzed and sorted on a FACSAria cell sorter (BD Biosciences, San Jose, CA, USA, http://www.bdbiosciences.com) with blue laser excitation (488 nm).

To characterize CPMV binding the cells were incubated with CPMV-AF488 for 3 hours on ice, washed, stained and analyzed by FACS.

To determine specific versus nonspecific binding, the cells were incubated with the unlabeled CaIX-P1 peptide at concentrations varying from 10−4 to 10−10 mol/L.

To avoid nonspecific binding, the cells were incubated in PBS containing 2% BSA for 30 min at room temperature before being stained with the corresponding primary Ab overnight at 4°C.

After Ab binding, the cells were warmed at 37°C to allow PrPC internalization for different indicated times (chase), the monolayers were fixed with paraformaldehyde (PFA) and incubated with the anti-mouse FITC-conjugated secondary antibody to label surface PrPC.

After primary antibody binding the cells were washed three times with ice-cold PBS-G and then resuspended in a 1.5 ml Eppendorf tube in 1 ml of ice-cold PBS-G containing 1∶200 Alexa 488-conjugated goat anti-rat IgM (Invitrogen, A21212) and incubated for 45 minutes in the dark at 4°C with gentle rocking.

In the live cell binding method, the cells were incubated with peptides in cell growth medium for two hours at 37°C.

After the binding assay the cells were washed with TBS (pH 7.4) and fixed with 4% PFA for 15 min at RT.

After adding 400  μl of 1 × binding buffer, the cells were analyzed by flow cytometry using a FACSAriaIII (BD Biosciences).

For the ligand-binding experiments, the cells were incubated with the required concentration of fluorescent ligand for 10 min at 37°C before imaging.

Biotinylated mouse monoclonal anti-human IgM antibody (Miltenyi Biotec, Clone PJ2-22H3 (isotype: mouse IgG1, intact molecule)) was also used when IgM bindings to the cells were checked.