Anti mouse igg a488

CHO-K1 cells were detached with 4 mM EDTA at 37 °C and harvested in PBS. Cells (3x10⁵ per experimental point) were incubated for 30 min on ice with 250 nM of the indicated viral recombinant proteins. Cells were then extensively washed with FACS buffer (PBS, 0.01% sodium azide and 0.5% bovine serum albumin) and incubated for 30 min at 4 °C with monoclonal anti-V5 antibody (Invitrogen) diluted 1:500 followed by anti-mouse IgG-A488 (Molecular Probes) diluted 1:500 in PBS. Finally, cells were resuspended in 0.5 ml FACS buffer and analysed in a FACSCalibur flow cytometer (BD Sciences).

HeLa or CHO-K1 cells seeded onto glass coverslips in 24-well plates were incubated with 250 nM of purified recombinant viral proteins for 30 min at 4 °C. Cells were then extensively washed with ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 12 min at room temperature. The membrane permeabilization step was omitted, thus after aldehyde quenching with 50 mM NH₄Cl for 5 min, cells were incubated with a monoclonal anti-V5 antibody diluted 1:500 (Invitrogen) followed by anti-mouse IgG-A488 (Molecular Probes) diluted 1:1000. Cell nuclei were stained with DAPI (Calbiochem). Images were acquired in a Leica DMI6000B automated inverted microscope equipped with a Hamamatsu Orca R2 digital camera.