Cell Surface Receptor Expression Profiling

Receptor expression was profiled essentially as previously described.16 (link) In brief, EOC cells (1.5 × 10⁵ cells per well in a 96-well plate) were seeded and washed in 200 μl of wash buffer (PBS/1% BSA) and incubated with 100 μl of wash buffer containing 1:500 of mouse anti-human monoclonal antibody against CAR (RmcB, Millipore, Watford, UK), 1:500 of CD46 (MEM-258, Abcam, Cambridge, UK) or mouse IgG control antibody (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 hour on ice. Cells were washed three times and incubated with a 1:500 dilution of goat anti-mouse Alexa Fluor 647 antibody (Invitrogen, UK) for 1 hour on ice. Cells were fixed in 4 % paraformaldehyde (PFA) for a minimum of 10 min at 4 °C for flow cytometry. 2 × 10⁴ gated events were acquired in channel FL-4 on a BD Accuri C6 (BD Biosciences, USA) flow cytometer and data analysed in BD Accuri C6 software version 1.0.264.21 (Becton Dickinson, USA).