Figure 4.

Internalization of GD2-specific mAb and increase of GD2 in endosomes in human melanoma cells incubated with GD2-specific mAb 3F8. (A) HTB63 cells (2 × 10⁵/well) were seeded and grown on glass coverslips in flat bottom six-well plates and incubated with Alexa Fluor 488 labeled mAb 3F8 (50 μg/ml) (3F8*). Alexa Fluor 488 labeled mAb 763.74 (50 μg/ml) (Ctrl*) was used as an irrelevant antibody control. Following a 3 h incubation at 37°C in a 5% CO₂ atmosphere, internalization of mAb 3F8 was determined by overlapping the confocal microscopy images of green fluorescence (Alexa Fluor 488 labeled mAb 3F8), and red fluorescence (endosomal marker, Rab 5). The results presented are representative of those obtained in two independent experiments. (B and C) HTB63 cells were incubated with mAb 3F8 (3F8) (50 μg/ml). CSPG4-specific mAb 763.74 (50 μg/ml) was used as a specificity control (Ctrl). Following a 3 h incubation at 37°C in a 5% CO₂ atmosphere, cells were harvested and 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). The results presented are representative of those obtained in three independent experiments (B). 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).