Hi Jahnu, you could try loading the ab peptide fibrils with thioflavin-T before adding it to cells. Although fluorescence is not quantitative, you could do a standard curve of ThT and fibrils.

The standard way of determining internalized ligands is to trypsin treat cells and measure the ligands that are trypsin insensitive after pelleting and washing the treated cells.

Thank you Steingrimur Stefansson . However I think once inside the cell the ThT molecules will bind to other proteins/peptide (containing ThT binding domain) and thus the specificity of the experiment will be compromised.

Regards,

Jahnu

You can check https://en.wikipedia.org/wiki/HaloTag

Alternatively: https://en.wikipedia.org/wiki/Click_chemistry and kits like these ones https://www.thermofisher.com/mx/es/home/life-science/cell-analysis/labeling-chemistry/click-chemistry-labeling-and-detection/click-chemistry-tools-for-biological-assays.html

Thank you S. K Sharma

Hi Jahnu, you could always do the classical internalization assay using radiolabeled I125, C14 or H3 ligand.

FlowCy and confocal are subjective for quantitatively determining internalization. Plus, fluorescence is not quantitative by itself because it does not follow Beer's law. Using radiolabeled ligands gives you an absolute quantitative value.