Nis elements viewer version 3

Live parental cells were incubated with MitoSOX Red (5 μM; Invitrogen, Carlsbad CA) for 30 min, washed and recovered in phenol red-free DMEM with 2% FBS and treated with docetaxel (50 nM). Equivalent exposure times were used at each interval for immunofluorescent microscopy, which was compensated for autofluorescence. Images were captured on a NIKON Eclipse TI-U microscope with a 20x ELDW or 10x or 40x Plan-Apo objective lens(Nikon) and NIS Elements Viewer version 3.22 (Nikon) imaging software. A negative control (non-transfected) cell subset was used to normalize background and autofluorescence following docetaxel treatment. Individual cells were tracked over time and cell death was assessed with the 2 TdT-Fluor In Situ apoptosis detection kit (Trevigen). Apoptosis induction (or loss of adherence to culture dish, which was used as an indicator of cell death) and changes in MitoSOX fluorescence were plotted for individual cells over time.

DNCCs or DTCCs were generated as described above and plated in eight chamber glass slides (BD Biosciences, San Jose, CA) at a concentration of 10⁵ cells/mL. Before lipid raft analysis, cells were first exposed to fluorescent lipids (NBD-PC or NBD-cholesterol; fluorescent in the green spectrum) at the indicated concentrations. Subsequently, lipid rafts were labeled by Vybrant^™ Alexa Fluor^™ 594 Lipid Raft Labeling Kit according to manufactures protocol (ThermoFisher Scientific). Briefly, cells were washed with PBS and CT-B (Cholera toxin subunit B) has been added with basal media (2 μg/mL) and incubated for 1 h at 4 °C. Cells were washed 3 times with cold PBS and anti CT-B (5 μL/mL in basal media) has been added to that. After incubation for 30 min at 4 °C, the cells were fixed with 4% paraformaldehyde for 20 min. Cells were stained with 4’,6-diamidino-2-phenylindole (DAPI) and imaged by fluorescence or confocal microscopy. Fluorescent images were obtained using three channels on an NIKON Eclipse TI-U microscope with a 20×ELDW, 10 or 40× Plan-Apo objective lens (Nikon, Melville, NY). NIS Elements Viewer version 3.22 (Nikon, Melville, NY) software was used to capture the images to file.