Example 4
Cell Binding Experiments
Cell lines. EBC-1 and CHO-K1 cells were cultured in DMEM with 4 mM L-glutamine (Sigma-Aldrich) and DMEM-F12+GlutaMax™ (Gibco®), respectively, both supplemented with 10% fetal bovine (Sigma-Aldrich) at 37° C. and 5% CO2.
Cell binding experiments. Cell binding experiments were performed by using EGFR-overexpressing EBC-1[3] and EGFR-negative CHO-K1 cells in combination with confocal fluorescence microscopy and flow cytometry, respectively. Washing and incubation steps were performed at 4° C. using PBS with 1% BSA. For microscopy based experiments, cells were grown on glass coverslips followed by consecutive labelling with 100 nM of respective antibody-conjugates and 1:200 diluted Streptavidin Alexa Fluor® 488 conjugate (Life Technologies). Then, cells were fixed with 4% paraformaldehyde, mounted with ProLong® Diamond Antifade Mountant with DAPI (Life Technologies) and scanned using a Leica TCS SP5 confocal microscope equipped with a 100× objective (Leica Microsystems). For flow cytometry, 2×105 cells were incubated with 100 nM of respective antibody-conjugates and in a consecutive step with 1:200 diluted Streptavidin Alexa Fluor® 488 conjugate (Life Technologies). Cell fluorescence was determined using a BD Influx cell sorter and BD FACS Sortware with detection of 2×104 events.