Bsa stain buffer

HUVECs were harvested with trypsin–EDTA. All immunofluorescent labeling and washing was performed in Stain Buffer (BSA) (BD Biosciences). Cells were incubated with an irrelevant isotype control or the mouse antihuman CD34 antibody (5 µg/mL, QBEND/10) for 1 h at 4 °C. After 3 times washing with cold Stain Buffer, FITC-conjugated goat antimouse IgG (1:100, Jackson ImmunoResearch) were added to the cells for 45 min in the dark at 4 °C, followed by washing. Flow cytometry analysis was performed using a FACSCalibur system (BD Biosciences) in combination with CellQuest software (BD Biosciences).

A total of 6×10⁴ cells were seeded in each well of an 8-well chamber slide (Millicell EZ slide, Millipore) and cultured overnight to allow adherence of the cells. The cells were fixed with 4% formaldehyde on ice for 20 minutes. After three washes, the cells were blocked with 0.2% (w/v) BSA stain buffer (BD Bioscience Pharmingen, San Diego, CA, USA) at room temperature for one hour. The cells were incubated with 100 μL of cetuximab and SPIONs diluted in 0.2% BSA stain buffer at the indicated concentrations on ice for one hour. Cells treated with 0.2% BSA stain buffer alone served as a negative control. After three washes, Dylight 488-conjugated goat F(ab′)₂ anti-human Fcγ secondary antibody diluted in 0.2% BSA stain buffer was added to detect the bound cetuximab. After a one-hour incubation at room temperature, the cells were stained with 300 nM DAPI nucleic acid staining solution (Molecular Probes, Life Technologies Co) and rinsed several times with 1× PBS. After mounting with ProLong^® Diamond antifade mountant (Molecular Probe), the slides were observed using a fluorescence microscope with the appropriate filters.