Mortensen Templeton lavede en statusopdatering 1 uge, 2 dage siden
Another limitation of this study is that the exact locus of APOE expression could not be examined in detail using a standard epifluorescence microscope in this study.Highresolution microscopy techniques would have been more ideal to identify the accurate loci of APOE expression and overcome the challenges of imaging densely packed cells at the earliest stages of neural induction. Primary antibodies were incubated at C overnight followed by washings with TBS.Secondary antibodies conjugated with fluorescent dyes were incubated at room temperature for hours followed by washings with TBS.Imaging was done with IX inverted epifluorescence microscope. Although the primary function of APOE is lipid transport, its expression is also found in other cell types outside the context of lipid metabolism in the brain. Furthermore, neuronal APOE can also be expressed at high levels under stress conditions such as brain injury although APOE expression is normally low in healthy neurons. The authors address this important question through use of human induced pluripotent stem cells and monitor APOE status throughout neural differentiation.The gene expression data presented here is clear and the changes in APOE are quite dramatic but the immunostaining is rather descriptive and is not quite as convincing.Can these be improved at all. In the updated manuscript, these images are shown in a separate figure. The PDF version should enable sufficient magnification to view the composite panels and clearly demonstrate more intracellular localisation on D cells.While the authors confirm that the ICC experiments were conducted for APOE on D cells, the data were not included in the manuscript due to the following reasons.According to the Targetmol’s Candesartan Cilexetil differentiation protocol, the cells were maintained at high density approaching near confluence from D to D.We observed that this inadvertently diminishes the quality of immunocytochemistry images for D cells, since clear boundaries of nuclei could not be easily identified with epifluorescence microscopy and further complicated the downstream quantification process.The possibility of dissociating D cells and plating them on to a different surface for better image quality and quantification was considered briefly.However, such additional handling was not done to the cells so that any potential source of artefacts that could mask the true state of D cells can be ruled out in our experiments.While the use of epifluorescence microscopy in our study can be seen as a clear limitation, APOE immunostaining patterns of D cells was not qualitatively different from that of D cells in our observations.Further investigations using threedimensional imaging techniques such as confocal microscopy will enable better imaging and quantification of densely packed cells on D.However, timedependent changes of various markers of differentiation would add further validity to our observations and unequivocally clarify whether APOE expression is indeed correlated with the differentiation state of the cells.Another limitation of this study is that the exact locus of APOE expression could not be examined in detail using a standard epifluorescence microscope in this study.The authors would like to thank the reviewer for the comment on the quantification of ICC images.We now include a quantification of the images in the updated manuscript.