Viable cell flow cytometry on human tumor cell lines was performed essentially as detailed elsewhere (Mitjans et al., 1995 (link)). Cells were harvested from culture using trypsin (0.5 µg/ml)/EDTA (0.2 µg/ml), which did not affect expression of the integrins, and washed in FACs saline buffer (PBS; 0.9 mM CaCl2; 0.5 mM MgCl2; 0.5% w/v BSA). They were then incubated with antibody diluted in FACs buffer (1 µg/ml; 60 min; 4°C), washed and stained using Alexa488 labeled goat-anti-rabbit IgG (Invitrogen, Karlsruhe, Germany) (30 min; 4°C). Finally, cells were re-washed and subjected to flow-cytometry collecting 20000 events. Murine anti-integrin antibodies were used under identical conditions (but at 10 µg/ml). The EM00212 antibody against the β3 cytoplasmic domain does not recognize its epitope on viable cells, and was used as the isotype matched RabMab control. The mean intensity of fluorescence (MIF) was expressed as the ratio to the MIF of the negative control (cells stained with PI, with an isotype matched control, and secondary labeled antibody).
For detection of LIBS epitopes, cells were washed and suspended in FACS saline buffer and then incubated for 15 min with various concentrations of cyclic RGD peptide, reactive with integrins αvβ3 and αvβ5 (Goodman et al., 2002 (link)). Cells were then incubated with RabMabs in the presence of the peptide; the washing, staining, and flow-cytometry procedure was done as described above.
For detection of LIBS epitopes, cells were washed and suspended in FACS saline buffer and then incubated for 15 min with various concentrations of cyclic RGD peptide, reactive with integrins αvβ3 and αvβ5 (Goodman et al., 2002 (link)). Cells were then incubated with RabMabs in the presence of the peptide; the washing, staining, and flow-cytometry procedure was done as described above.
