Cfda se cell tracer kit

Cell adhesion and proliferation were studied by confocal imaging (Leica TCS SP2 confocal microscope). A co-seeding of hOBs and hACs (6 × 10⁴ cells) was performed on each scaffold sample. Both cell lineages were cultured in the chondrocyte medium described above. Before to co-seeding, hOBs nucleus and hACs cytoplasm were marked with Höechst (Höechst 33342 trihydrochloride solution, Invitrogen) and Vibrant (CFDA SE cell tracer kit, Invitrogen) staining, respectively. Briefly, the Höechst staining solution was added to hOBs cell solution at a concentration of 0.5 µg/mL, incubated at 37 °C for 15 min, followed by centrifugation and the pellet washed with PBS. After that, 7 µL of hOBs were seeded on the porous section of each scaffold and subsequently incubated at 37 °C and 5% CO₂ for 40 min. Afterwards, 200 µL of culture medium were added and cultured for 24 h for hOBs adhesion. After this pre-culture, Vibrant probe was added at 0.5 µg/mL to hACs cell solution, and the mixture was incubated at 37 °C for 15 min in dark conditions. The solution was centrifuged, and a pellet was washed with PBS three times. After that, 7 µL of hACs were seeded on the hydrogel section of each scaffold and subsequently incubated at 37 °C and 5% CO₂ for 40 min. Afterwards, 150 µL of culture medium were added and cultured for cell viability studies.