Goat anti mouse igg r pe

SS1P was purified in our laboratory as described previously (30 (link)). Clinical grade RG7787 was manufactured by Roche Innovation Center, Penzberg, Germany. Alexa-labeled RG7787 was made using an Alexa labeling kit from Invitrogen. HB21(Fv)-PE40 immunotoxin was made in our lab. Anti-MSLN (mouse) was purchased from Rockland Immunochemicals (code# 200-301-A88), GAPDH (rabbit mAb 14C10; Product #2118) from Cell Signaling Technology, Goat anti-mouse IgG-HRP antibody from Santa Cruz Biotechnology (Cat. # sc-2004)), Goat anti-mouse IgG-R-PE from Jackson Immunoresearch (Code: 115-116-146), QuantiBrite PE Beads from BD Biosciences, and Cell counting kit-8 from Dojindo Laboratories.

To quantify the cell surface mesothelin expression, cells were grown for 2 days, harvested, washed with FACS buffer (PBS with 5% FBS and 0.1% NaN₃), and incubated with 5 μg/mL of mouse antimesothelin MN antibody^{23 (link)} (Rockland Immunochemicals Inc., Gilbertsville, PA) on ice for 30 minutes. After washing, cells were incubated with goat antimouse IgG-R-PE (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) on ice for 30 minutes in the dark, and washed again twice. Mesothelin expression was analyzed on a FACSCalibur. QuantiBRITE PE beads (BD Pharmingen) were used to quantitate the number of mesothelin sites per cell. To evaluate RIT internalization and the effect of ABT-737 on uptake, cells were incubated for 3 hours at 37°C with 2 μg/mL of SS1P-Alexa647 alone or in combination with 10 μM ABT-737. SS1P was labeled with the Alexa Fluor 647 Labeling Kit (Invitrogen) according to manufacturer’s instructions. After incubation, cells were washed with FACS buffer, and surface-bound SS1P was removed by stripping cells with glycine buffer (0.2 mol/L glycine, pH 2.5 and 1 mg/mL of BSA) for 10 minutes, neutralized with Tris-HCl 1M pH 8, and washed with FACS buffer. Fluorescence intensity was analyzed on a FACSCalibur.