The viability of ECs and SCs in both mono- and cocultures was studied at d 1, d 7, and d 14 time points with qualitative live/dead viability staining as described previously [13 (link), 16 (link)]. The samples were incubated at 0.5 mM CalceinAM (green fluorescence; Molecular Probes) and 0.25 mM EthD-1 (green fluorescence; Molecular Probes) in DBPS (Sigma-Aldrich) for 45 min at RT and imaged with a fluorescence microscope (Olympus IX51S8F-2; camera DP71, Tokyo, Japan). The viable cells were stained green, and the dead cells were stained red. The background fluorescence caused by the scaffold without cells was used as a negative control.
The CyQUANT™ cell proliferation assay kit (Invitrogen) measuring the total DNA amount was used to evaluate the proliferation of ECs and SCs in monocultures at d 1, d 7, and d 14 as previously described[16 (link)]. Briefly, the cells were lysed with 0.1% Triton-X-100 buffer (Sigma-Aldrich) and stored at − 70 °C until analysis. After the freeze–thaw cycle, the working solution containing CyQUANT™ GR dye and cell lysis buffer was added, and the fluorescence at 480/520 nm was measured with a Victor 1420 Multilabel Counter microplate reader (Wallac, Turku, Finland).
The CyQUANT™ cell proliferation assay kit (Invitrogen) measuring the total DNA amount was used to evaluate the proliferation of ECs and SCs in monocultures at d 1, d 7, and d 14 as previously described[16 (link)]. Briefly, the cells were lysed with 0.1% Triton-X-100 buffer (Sigma-Aldrich) and stored at − 70 °C until analysis. After the freeze–thaw cycle, the working solution containing CyQUANT™ GR dye and cell lysis buffer was added, and the fluorescence at 480/520 nm was measured with a Victor 1420 Multilabel Counter microplate reader (Wallac, Turku, Finland).
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