Analysis of DOX uptake by fluorescence microscopy and flow cytometry.
A) Observation of cellular uptake of apatite-DOX by fluorescence microscopy in SW480 cells. Cells were grown (5×105 cells/dish) on 60 mm culture dishes. After 1 day, the medium was replaced by 1 ml of fresh medium and free DOX or DOX-loaded apatite particles (200 nM DOX equivalents) were added. After 4 h, the culture dishes were washed 3 times with PBS and the extracellularly bound particles were removed by 5 mM EDTA in PBS. Scale bar, 200 μm. B) Analysis of cellular uptake of apatite-DOX by flow cytometry in NIH:OVCAR-3 cell line. The cells were seeded (0.5×106cells/dish) in a 60 mm tissue culture dish, incubated overnight. 5 ml of DOX-loaded particles prepared with 200 nM DOX concentration in medium was introduced to each well and incubated for 4 h. The cells were then trypsinized, washed three times with PBS solution and then fixed with 10% formaldehyde. After filtering through a nylon mesh, cell fluorescence was measured by flow cytometry.
