Qifikit

To quantify surface mesothelin molecules, adherent cells were washed with DPBS, coated with Versene and incubated at 37°C for ~15 min. After incubation, flasks were tapped vigorously to promote complete detachment. The cells were diluted with DBPS at 1:2 ratio (Versene:DPBS) and counted. A total of 300,000 cells were centrifuged at 500×g for 5 min. Cells were resuspended in 300 µL 1× FACS buffer (DPBS +1% BSA). One hundred microliters of aliquot was divided into two v-bottom wells. Cells were washed one more time with 100 µL FACS buffer, then stained with 100 µL of anti-MSLN antibody at 10 µg/mL (R&D Systems, clone 618923) on ice for 30 min. After the primary stain, 50 µL of calibration beads from the QIFIKIT (Agilent) were added. The mixture was washed with 100 µL FACS buffer twice, then stained with 100 µL of anti-mouse F(ab′)2-Goat Alexa Fluor 647 (Invitrogen) antibody diluted 2000× in FACS buffer for 45 min on ice. The stained cells and beads were washed 2× with 100 µL FACS buffer, then resuspended in 100 µL FACS buffer to measure fluorescence. The calibration curve plotting median fluorescence intensity of each bead population versus number of molecules was plotted according to the manufacturer’s protocol provided by the QIFIKIT (Agilent). The number of endogenous MSLN molecules was determined using the cell’s MFI and the calibration curve generated with the QIFIKIT beads.