Cell titer blue

CellTiter Blue® (Promega, Madison, WI) is used for the assessment of cell viability according to the manufacturer’s instructions. 5000 cells/well are seeded in the 96–well cell culture plates and incubated at 37°C for 24 h in a 5% CO₂ incubator. Cells are then treated with the various formulations (free drug, polymer drug conjugate alone and targeted polymer drug conjugates) in complete DMEM medium for 48 h. For the targeted treatment, SKOV3 cells were treated with anti-HER2/neu affibody X anti-DTPA Fab bispecific complex (10, 20 or 40 μg/ml) and MCF7 cells were treated with Biotinylated anti-DTPA bsMAbCx (10, 20, or 40 μg/ml) for 1 h. After pre-treatment, media is removed and the plate is washed 2X with 200 μL of complete medium. Then, Ptxl or Dox, D-Ptxl-PGA or D-Dox-PGA at 0.001 to 15 μg/ml is added to the medium. The drug treated cells are incubated at 37°C for 48 h. The cells are then washed and incubated with 50 μL of 1:5 dilution of CellTiter Blue® reagent for 2 h. Cell viability is evaluated by measuring the fluorescence (excitation 530 nm, emission 590 nm) using a Synergy HT multi-21 detection microplate reader (Biotek, Winooski, VT). Cells treated with complete media alone are used as a control to calculate 100% cell viability. The studies are performed in triplicates at 3 different occasions.