Flow cytometry analysis allowed measuring the expression of Her2 on the surface of PC-3 cells [28 (link)].
As previously described [29 (link)], QuantiBRITE PE (BD Biosciences, San Jose, USA) was used to estimate the absolute number of Her2 receptors on cell membranes. About 100,000 cells of PC-3 were incubated under saturated conditions with PE Mouse Anti-Human HER-2/neu (BD Biosciences, San Jose, USA) for 30 min at 4 °C before being rinsed with PBS. IgG2a-PE anti-mouse antibodies (Fisher Scientific, Illkirch, France) were used for isotopic control. Analysis was then immediately performed on Gallios Beckman Coulter. Assuming our anti-HER-2 PE antibody has a 1:1 fluorochrome/antibody ratio, PE median fluorescence intensity (MFI) was measured for all cell lines and reported on a log–log graph with MFI vs. PE molecules, after subtracting isotopic control MFI. All the measures were performed in triplicate.
As previously described [29 (link)], QuantiBRITE PE (BD Biosciences, San Jose, USA) was used to estimate the absolute number of Her2 receptors on cell membranes. About 100,000 cells of PC-3 were incubated under saturated conditions with PE Mouse Anti-Human HER-2/neu (BD Biosciences, San Jose, USA) for 30 min at 4 °C before being rinsed with PBS. IgG2a-PE anti-mouse antibodies (Fisher Scientific, Illkirch, France) were used for isotopic control. Analysis was then immediately performed on Gallios Beckman Coulter. Assuming our anti-HER-2 PE antibody has a 1:1 fluorochrome/antibody ratio, PE median fluorescence intensity (MFI) was measured for all cell lines and reported on a log–log graph with MFI vs. PE molecules, after subtracting isotopic control MFI. All the measures were performed in triplicate.
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