![Cell internalization analyzed by flow cytometry. (A,C) Trypsinization to quantify internalized mAbs. Different forms of mAbsCy5.5 bound to the MS1 cells at 4°C. After washing out the unbound mAbs, internalization was induced for 2 hours at 37°C. To detect only internalized mAbs, cell surface proteins were trypsinized. The remaining cellular fluorescence was then analyzed by flow cytometry. (A) Anti-Robo4 mAbsCy5.5, (C) Anti-VEGFR2 mAbsCy5.5. Black, nontrypsinized group; gray, trypsinized group; white, negative control (anti-His[scFv]Cy5.5, anti-His[dscFv]Cy5.5, or anti-FLAG[IgG]Cy5.5). (B,D) Time course of the internalization. After binding at 4°C, internalization was induced for 0.5, 1, 2, 4, or 8 hours at 37°C. The ratio of internalization was calculated using the following formula: internalization (%) = {internalized mAb}/{total bound mAb} × 100 (%) = {(MFI of mAb)T – (MFI of negative control)T}/{(MFI of mAb)N – (MFI of negative control)N} × 100 (%). T, trypsinized group; N, nontrypsinized group; MFI, mean fluorescence intensity. (B) Closed and open markers indicate R4-13i and R4-16, respectively. (D) Closed and open markers indicate V2-05i and V2-02, respectively. (B,D) Circles, diamonds, and squares indicate scFv, dscFv, and IgG, respectively. Each experiment was performed in triplicate. Values are shown as means ± SD. **P < .01; internalizing mAb versus low-internalizing mAb in each form by 2-way ANOVA (n = 3).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/121/14/10.1182_blood-2012-12-468363/4/2804f3.jpeg?Expires=1720807354&Signature=vcsco5nNcCRzGNN2JjLjSkWwuon29o2WRnf2w186qDA6qTSoU4NZ1jw8xlFp-mJ9ZyXZ5VaXjlBn-sqq-kwzg1L-3WLKqiEIfJe4UxGlNRLx3cCqmgpvtJ0tLPdlxasZf48DqVy~bji94kkfJ4omy63unZwXMXdzbMpvKqCRzDlFOF2Vl6Rpf4xeM1plU3RNWoWex0DVPawaomUuJblS5UCG5yXWS2s2VI3NK28RvmYwbepv5Hq7QQT8XatGjfgze5COfmOS61AxbV5~XUwPh9vPPT75OMto6mySspcHw7X9pqxxaG0cqhC91CjWGtrTBY-xXUM4sCkhzedM2uDjnA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Cell internalization analyzed by flow cytometry. (A,C) Trypsinization to quantify internalized mAbs. Different forms of mAbsCy5.5 bound to the MS1 cells at 4°C. After washing out the unbound mAbs, internalization was induced for 2 hours at 37°C. To detect only internalized mAbs, cell surface proteins were trypsinized. The remaining cellular fluorescence was then analyzed by flow cytometry. (A) Anti-Robo4 mAbsCy5.5, (C) Anti-VEGFR2 mAbsCy5.5. Black, nontrypsinized group; gray, trypsinized group; white, negative control (anti-His[scFv]Cy5.5, anti-His[dscFv]Cy5.5, or anti-FLAG[IgG]Cy5.5). (B,D) Time course of the internalization. After binding at 4°C, internalization was induced for 0.5, 1, 2, 4, or 8 hours at 37°C. The ratio of internalization was calculated using the following formula: internalization (%) = {internalized mAb}/{total bound mAb} × 100 (%) = {(MFI of mAb)T – (MFI of negative control)T}/{(MFI of mAb)N – (MFI of negative control)N} × 100 (%). T, trypsinized group; N, nontrypsinized group; MFI, mean fluorescence intensity. (B) Closed and open markers indicate R4-13i and R4-16, respectively. (D) Closed and open markers indicate V2-05i and V2-02, respectively. (B,D) Circles, diamonds, and squares indicate scFv, dscFv, and IgG, respectively. Each experiment was performed in triplicate. Values are shown as means ± SD. **P < .01; internalizing mAb versus low-internalizing mAb in each form by 2-way ANOVA (n = 3).
