5.2. Cell Culture, SRB Cytotoxicity Assay, Preparation of Cell Monolayers

Cell lines 1411HP (germ cell tumor), A2780cis (ovarian carcinoma) and HT29 (colorectal carcinoma) were cultivated with RPMI medium (containing 10% fetal bovine serum and 10% penicillin/streptomycin) at 37 °C/5% CO₂ in a humid atmosphere.

For the sulforhodamine-B (SRB) cytotoxicity assays, cells were seeded in 96-well plates and incubated for 24h. The pHPMA-Dox-Cy7 conjugate was pre-incubated for 24 h in phosphate buffers with different pH values ranging from 5.5 to 7.4. Afterwards, the stock solution (equivalent to a doxorubicin concentration of 1.44 mM) was used directly to prepare serial dilutions (0.001 µM–10 µM) which were added to the cells. After incubation/treatment for 2 h, the supernatant was removed and fresh RPMI medium was added to the cells for another incubation period of 96 h. All following steps were performed according to the SRB assay protocol previously described [13].

For the microscopic examination of fixed monolayer cells, the respective tumor cells were seeded and cultivated in chamber slides. After 24 h, they were incubated with pHPMA-Dox-Cy7 (equivalent to a Dox concentration of 30 nM) for 8 h. Then, the supernatant was removed; the cells were rinsed with PBS and were formalin-fixed. After washing with PBS, the cells were treated with Alexa Fluor^® 488 Phalloidin to stain the cytoskeleton. Finally, the cell monolayers were rinsed and preserved using mounting medium to be analyzed using multispectral fluorescence microscopy.