Quantification of cellular uptake by flow cytometry

A549 cells, HUVECs, RAW264.7 cells, and H9C2 cells were cultured in 12-well plates at a density of 2 × 10⁵ cells per well in 1 mL of culture medium for 12 h. Then H9C2 cells were treated with fresh medium with or without 2 μM doxorubicin (DOX), while A549 cells, HUVECs, and RAW264.7 cells were incubated without DOX. After 24 h, cells were incubated with 1 mL of medium containing 50 μg Cy5-labeled NPs. Then cells were incubated for different periods of time, followed by flow cytometry analysis (Accuri C6, BD Biosciences). Similarly, cellular uptake was studied after incubation with different doses of Cy5/TPCD NP (varying from 1, 5, 10, 50, to 100 μg/mL) for 2 h.

In another experiments, cellular uptake profiles of TPCD NP or TTPCD NP in H9C2 cells were compared. In this case, H9C2 cells were treated with 2 μM DOX. After 24 h, cells were incubated with 1 mL of medium containing 50 μg Cy5-labeled NPs for different time periods, and then analyzed by flow cytometry analysis.