Endocytosis Assay in MSC80 Cells

Endocytosis was tested in MSC80 cells using the calcofluor white (CW) assay. CW is a fluorescent dye known to bind with high affinity to cellulose. Briefly, cells were incubated for 2 h with both CNC/CD and CW in the presence or absence of endocytosis inhibitors dynasore (dynamin-mediated endocytosis inhibitor; 20 μM), or genestein (caveolae-mediated endocytosis inhibitor; 50 μM). Cells were then washed with Hanks’ balanced salt solution and incubated with a basic buffer (KOH+Hanks’ balanced salt solution) that promotes cell detachment and allows the release of intracellular fluorescent complexes. The entry of CW/CNC/CD complex into the cell was assessed by the relative fluorescence intensity using a Fluoroskan plate reader (Ex/Em = 355/433 nm).