Ctr 6500 confocal microscope

Epithelial cell sheets were fixed with 4% paraformaldehyde and washed in PBS. The Costar filter inserts were cut from the plastic support, placed onto a microscope slide, and mounted with Vectashield Mounting Media with DAPI. Using a Leica CTR6500 confocal microscope, images of six random fields from each filter were captured at ×20 magnification. Manual counts were performed by enumerating the blue nuclei and GFP⁺ cells. All epithelial cells within these six fields were counted. For determining the percentage of positive cells, the numerator was the number of GFP⁺ cells and the denominator was the total number of cells in the six ×20 magnification fields we counted for each epithelial sheet.

Cells on glass coverslips were treated as described above, fixed with 4% formaldehyde, and washed twice with phosphate-buffered saline (PBS) before blocking was performed for 30 min in PBS containing 1% Triton X-100, 2% bovine serum albumin (BSA), and 0.02% sodium azide. Primary antibody dilutions were made in same block buffer, and cells were incubated overnight at 4°C. Following two PBS washes, secondary antibody treatment was done for 1 h at 4°C. Coverslips were then mounted to glass slides with 15% (vol/vol) glycerol–PBS. Images were taken with a Leica CTR 6500 confocal microscope, and data were quantified using the granularity module in the MetaXpress software suite (Molecular Devices).

The cells were seeded on a cell culture 8-well glass slide (50000 cells per well) followed by an overnight incubation. Then the cells were incubated with the DOX derivative solutions (100 µM, 250 µl per each well) in serum free medium in the CO₂-incubator for 30 min. The cells were additionally stained with Hoechst 33342 (50 µM, 10 min) and Calcein AM (25 µM, 15 min) for nuclei and cytoplasm visualization, respectively. In some experiments, in order to visualize mitochondria, the cells were stained with MitoTracker Orange (500 nM, 30 min). Finally, the cells were washed three times with PBS, mounted in the CC/Mount fluorophor protector, and observed using Leica CTR 6500 confocal microscope (Germany). The excitation wavelengths were 360 nm for Hoechst, 488 nm for Calcein AM, and 543 nm for DOX derivatives or MitoTracker Orange. Fluorescence signals were collected at 380-460 nm for Hoechst, 500-530 nm for Calcein AM, and 560-650 nm for DOX or MitoTracker Orange. The images were processed in Image J software.