the cell membrane, z-series confocal images of PC12 cells incubated with F-Ab40 and AF633-Trf for 60 min were obtained and presented as XY, XZ, and YZ projections. The endocytotic marker used in these studies, AF633-Trf, localizes primarily in early endosomes. Therefore, similar uptake studies were conducted using Dil labeled low density lipoprotein complex to capture the accumulation of F-Ab40 in the late endosomes. These studies also demonstrated a partial co-localization of F-Ab40 with Dil-LDL at both 15 and 30 min following incubation. PC12 cells incubated with 15 mg/ml F-Ab40 and 5 mg/ml Alexa Fluor 647 cholera toxin, a marker for caveolae mediated endocytosis, exhibited punctuate localization of F-Ab40 but AF647-CT fluorescence diffused throughout the perinuclear region. Both fluorophores co-localized very slightly. The uptake of AF633-CT in PC12 showed significantly greater uptake of F-Ab40 than the normal cells at 37uC. However, no detectable AF633-Trf signal was observed in these cells. Based on the DIC and fluorescence image composite, the cells depleted of ATP appeared normal at the end of the experiment. The results from flow Oritavancin (diphosphate) site cytometry studies conducted on ATP depleted cells were in agreement with the observations made in the microscopy studies. In the PC12 cells depleted of cellular ATP, the uptake of F-Ab40 at 37uC was significantly higher than that of the normal cells. The uptake of AF633-Trf, on the other hand, was significantly impaired in ATP depleted cells. These results clearly demonstrated that the PC12 cells simultaneously treated with F-Ab40 and AF633-Trf at 37uC accumulated both the fluorophores without noticeable colocalization. However, at 4uC or under ATP depleted conditions, which inhibit active transport processes including receptor mediated endocytosis, F-Ab40 internalization by PC12 cells was not inhibited whereas the receptor mediated endocytosis of AF633-Trf was substantially impaired. Role of endocytosis in the internalization of F-Ab42 by differentiated PC12 cells Following the incubation with F-Ab42 
and AF633-Trf, the PC12 cells internalized both proteins in the perinuclear region. A Composite of green and red channel images demonstrated little co-localization of the fluorophores, which is also confirmed by the magnified inset of the composite image. The histograms of cellular fluorescence obtained from 19478133 flow cytometry analysis demonstrated that the uptake of F-Ab42 at 4uC was not significantly different from that at 37uC; but the uptake of AF633-Trf at 4uC was significantly lower than the uptake at 37uC. Cellular Uptake of Ab Proteins 6 Cellular Uptake of Ab Proteins Uptake of F-Ab40 in rat primary hippocampal neurons Observations made in PC12 cells and adult hippocampal neurons were verified in RPH neurons. At 37uC, RPH neurons internalized F-Ab40 as well as AF633-Trf without significant co-localization. The uptake of AF633-Trf reduced significantly in the RPH neurons incubated at 4uC, whereas, the uptake of FAb40 22440900 was not affected. In RPH neurons depleted of cellular ATP, the uptake of F-Ab40 increased considerably, whereas no detectable uptake of AF633-Trf was observed. Uptake of F-Ab40 in bovine brain microvascular endothelial cells In contrast to the observations made hitherto in neuronal cells, BBME cells accumulated F-Ab40 in the acidic cell organelles stained with Lysotracker RedH. In addition, a comparison of fluorescence intensities in BBME cells treated simultaneously with F-Ab40 and AF633-
