Doxorubicin has an intrinsic fluorescence detectable in the visible spectral region and it can be efficiently excited with a blue light source [49 (link)]. For a quantitative estimation of doxorubicin accumulation, cells were seeded on laminin-coated 12-well plates at the density of 2 × 105 cells/well. The next day, cells were treated with doxorubicin (100 μM). After 1 h, cells were observed under a fluorescence microscope (Axioskop2, Zeiss, Oberkochen, Germany), using a 40× objective through the Axion Vision program (Zeiss, Oberkochen, Germany).
The culture media were then supplied with verapamil (100 μM) or N-8-Iper (1.5 μM for GB7 cells and 12.5 μM for G166 cells) where required. After 3 h, the treated cells were observed again, and representative fields were photographed by fluorescence microscope (Axioskop2, Zeiss, Oberkochen, Germany). The culture media were collected from each well and the fluorescence intensity was measured. At the same time, to assess the amount of doxorubicin accumulated into the cells, they were collected, and fluorescence was measured in the cell lysates. The fluorescence intensity for both media and lysates were measured by the GloMax Multi Detection System (Promega, Madison, WI, USA) (λex, 470 nm; λem, 590 nm). The lysates and the protein content were obtained as indicated below in the Western blot section.
The culture media were then supplied with verapamil (100 μM) or N-8-Iper (1.5 μM for GB7 cells and 12.5 μM for G166 cells) where required. After 3 h, the treated cells were observed again, and representative fields were photographed by fluorescence microscope (Axioskop2, Zeiss, Oberkochen, Germany). The culture media were collected from each well and the fluorescence intensity was measured. At the same time, to assess the amount of doxorubicin accumulated into the cells, they were collected, and fluorescence was measured in the cell lysates. The fluorescence intensity for both media and lysates were measured by the GloMax Multi Detection System (Promega, Madison, WI, USA) (λex, 470 nm; λem, 590 nm). The lysates and the protein content were obtained as indicated below in the Western blot section.