Calcein am green fluorescence

The cell attachment was verified by determining the DNA amount using CyQUANT Cell Proliferation Assay kit (Invitrogen). Briefly, 20 000 urothelial cells from one patient were seeded on to 3 parallel PLCL and PTMC membranes and cultured for 24 h. The cells were lysed with 0.1 % Triton-X-100 buffer (Sigma-Aldrich) and stored at -70 °C until analysis. The samples were thawed and 20 µl of each sample was mixed with 180 µl of working solution containing CyQUANT GR dye and lysis buffer. The fluorescence at 480/520 nm was measured with a multiplate reader (Victor 1420 Multilabel Counter; Wallac; Turku, Finland).
To verify the viability of human urothelial cells on PLCL and PTMC membranes, we used qualitative live/dead fluorescent staining. The urothelial cells from one patient, 30 000 cells/cm 2 , were seeded on to two parallel membranes and the viability of urothelial cells was verified after 1 and 2 weeks of cell culture as described before. 10, 11 Briefly, the cells were incubated at room temperature with a mixture of 0.25 µM calcein AM (green fluorescence, Molecular Probes, Waltham, USA) and 0.3 µM ethidium homodimer-1 (red fluorescence, EthD-1, Molecular Propes) for 30 minutes. A fluorescence microscope (Olympus, IX51S8F-2, camera DP71, Tokyo, Japan) was used to image viable cells (green fluorescence) and dead cells (red fluorescence).