Quantifying Mesothelin-Targeted Internalization

RITs were labeled with the Alexa Fluor 647 Labeling Kit (Invitrogen) for 3.5 hrs and purified according to manufacturer’s instructions. Harvested cells were incubated for 30, 75 and 150 min at 37°C with a saturating 2 μg/ml of SS1P-Alexa647 or RG7787-Alexa647 and processed as previously described [22 (link)]. Fluorescence intensity was analyzed on a FACSCalibur. Uptake was expressed in number of internalized RG7787 molecules, which was calculated by assuming the RG7787-Alexa647 geomean surface expression of KLM-1 equal to 60 x 10³ RG7787 molecules (= surface mesothelin binding sites per KLM-1 cell, as evaluated by flow cytometry and QuantiBRITE R-PE beads).