Mouse anti human trop2 antibody

Cells were stained with Mouse anti-human TROP2 antibody (BD Bioscience 551317, 1/200) in antibody staining buffer (0.9% (w/v), sodium azide and 2% FBS in PBS) for 1 hour. Following three washes in PBS, Alexa Fluor® 647 Goat Anti-Mouse IgG secondary antibody (Invitrogen A21236, diluted 1 in 1000 in antibody staining buffer) was incubated for one hour at 4°C. Stained cells were then washed three times in antibody staining buffer prior to FACS sorting and analyzing.
Cell sorting was performed using a MoFlo®Astrios^TM (Beckman Coulter). The gating strategy to define negative populations for ALDH and TROP2 staining was designed so as to include at least 99.9% of cells in the DEAB and isotype negative control samples. A distinct population highly expressing TROP2 was identified and sorted from PC3 cells (~2.75% of total viable cells). The same gating strategy was applied to LNCaP, DU145 and 22Rv1 cells. Since no individualized TROP^high population was detected in these cell lines, the TROP2^high population was defined as the top 2% of TROP2-expressing cells. FACS analysis was performed using a FACSCanto™ II (BD Science).

Slide-mounted cryo-cut tumor sections were fixed with 4% paraformaldehyde for 10 minutes and stained with mouse anti-human TROP2 antibody (BD Bioscience 551317, 1/200) overnight at 4°C followed by incubation with fluorescein horse anti-mouse IgG (Vector laboratories, 1/200) for 1 hour at room temperature. Hoechst 33342 (Invitrogen) was used to stain nuclei. Fluorescent images were taken with a Nikon digital camera attached to a Nikon A1R confocal microscope (Nikon).