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. 2015 Apr 2;4(8):e1023975. doi: 10.1080/2162402X.2015.1023975

Figure 5.

Figure 5.

Association of apoptosis induction with GD2-specific mAb internalization and increase in GD2-containing endosomes in human melanoma cells incubated with GD2-specific mAb 3F8. (A) HTB63 cells (2 × 105/well) were seeded and grown on glass cover slips in flat bottom six-well plates and incubated with Alexa Fluor 488 labeled GD2-specific mAb 3F8 (50 μg/ml) (3F8*). Alexa Fluor 488 labeled F(ab’)2 fragments of mAb 3F8 (50 μg/ml) (Ctrl-*) was used as a negative antibody control. Following a 3 h incubation at 37°C in a 5% CO2 atmosphere, internalization of GD2-specific mAb 3F8 was determined by overlapping the confocal microscopy images of green fluorescence (GD2-specific mAb 3F8) and red fluorescence (endosomal marker, Rab 5). The results presented are representative of those obtained in three independent experiments. (B and C) HTB63 cells were incubated with mAb 3F8 (3F8) (50 μg/ml). F(ab’)2 fragments of mAb 3F8 (50 μg/ml) were used as a negative antibody control (Ctrl). Following a 3 h incubation at 37°C in a 5% CO2 atmosphere, cells were intracellularly stained with Alexa Fluor 488 labeled GD2-specific mAb. Increase of GD2 containing endosomes (yellow color) in HTB63 cells was determined by overlapping the confocal microscopy images of green fluorescence (GD2-specific mAb) and red fluorescence (endosomal marker, Rab 5) (B). The results presented are representative of those obtained in three independent experiments. Overlapped confocal microscopy images were analyzed by ImageJ software for the increase of GD2 containing endosomes (yellow particles). Data are expressed as mean of GD2 containing endosomes per 100 cells ± SD of the results obtained in three independent experiments (C). (D) HTB63 cells were incubated with mAb 3F8 (50 μg/ml). Following a 24 h incubation at 37°C in a 5% CO2atmosphere, induction of apoptosis was determined by intracellular PI-staining. F(ab’)2 fragments of mAb 3F8 were used as a negative antibody control (Ctrl-). mAb HO-1 was used as an isotype matched control (Ctrl). The results presented are representative of those obtained in three independent experiments.