We have four fluorescently labeled endocytic markers including human serum albumin (HSA-Atto488) labeled in house, human transferrin (hTf-Alexa488) from ''Biozol Diagnostica'', Lactosylceramide (LacCer-BODPY FL) from ''Life-technology'' and 70 kDa and 10kDa Dextran-FITC from ''Sigma''. Only LacCer has taken up well by our cells in 1uM after 15 minutes at 37C, however, others were taken up very few even in very high concentration and even up to1 hour at 37C.

We tested:

- HSA from 5 to 500 ug/ml

- hTf from 5 to 500 ug/ml

- Dextran from 0.1 to 10 mg/ml

Our cells are cortical neurons differentiated for 8 days from neuronal progenitor cells (NPCs). All materials are fresh and fluorescently very bright.

We also tested 2 hours incubation with medium without N2 and B27 supplement before exposure to the markers, to somehow starve cells, but it did not work. We also find a similar problem with LUHMES differentiated for 6 days. I am using a fluorescence Plate reader along with a fluorescent microscope as well and FluoBrite DMEM as a media and BackDrop quencher for fluorescence measurements.

Is there any similar observation or suggestion? Could it be due to the immature innate of these neurons?! Are there other common markers of endocytic pathways to test?

I would appreciate your kind help in advance