Lysotrackerr red dnd 99

In this study, to track SPION instead of Dtxl, Coumarin 6, which serves as green fluorescent marker, was encapsulated. To determine the cellular localization of Coumarin 6 labeled SPION (C6-SPION), C4-2 and PC-3 cells were seeded at a density of 2×10⁴ on 8-well chamber glass slides (Nalgene Nunc Intl., Rochester, NY). Cells were then treated with 2 μg of Coumarin 6 equivalent C6-SPION for 3 hrs. After 2 hrs of incubation, cells were fixed using 2% paraformaldehyde for 10 min, permeabilized with 0.1% TritonX-100 in 1X PBS for 10 min, and washed twice with an additional 1X PBS, followed by a blocking step with 2% goat serum in PBS for 1 hr. Then cells were incubated with 30 nM Mito Tracker Red or 50 nM Transferrin from Human Serum, Texas Red^® Conjugate or 75nM LysoTrackerR Red DND-99 (Life Technologies) to stain as a marker for mitochondria, endosome, and lysosome, respectively. Finally, nuclei were counter stained with DAPI (4′,6-diamidino-2-phenylindole, Life Technologies) and mounted in Vectashield Mounting Medium (Vector Labs, Burlingame, CA). Finally, cells were visualized under a laser confocal microscope (Carl Zeiss LSM 710, Oberkochen, Germany) under 400X magnification using oil immersion objective.