Live-Dead Cell Staining Kit (BioVision)

Protocol tips

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	Mix 1 μL of Live-Dye and 1 μL of PI in 1 mL of Staining Buffer and incubate for 15 min at 37°C.

Protocol tips
Mix 1 μL of Live-Dye and 1 μL of PI in 1 mL of Staining Buffer and incubate for 15 min at 37°C.

Publication protocol

Five samples were prepared in every group for viability analysis. All samples were placed into a 70 °C water bath for 30 min to inactivate the residual mTG before the cell experiment. After that, the samples were immersed in ethanol (75%) for 12 h under UV radiation, and then the channels in the construct were washed with PBS three times. Finally, the construct was immersed in fresh RPMI 1640 medium for 6 h before injection. After trypsinization, a cell suspension with a density of 3 × 106 cells/mL was prepared in RPMI 1640 medium. Then, 10 mL of cell suspension was slowly perfused into the macrochannels in the constructs by syringe (groups Gel-F127-PCL and Gel-F127). After 4 h of cell attachment, the cell-laden constructs were statically cultured, with the medium changed every day in a 5% CO2 incubator. Live-Dead Cell Staining Kit (BioVision, Inc., San Francisco, CA, USA) was used to assess cell viability and distribution within the construct. This kit utilizes a cell-permeable green fluorescent dye, Live-Dye, to stain live cells. Dead cells can be easily stained using propidium iodide (PI), a cell nonpermeable red fluorescent dye. Staining Solution for the construct was prepared using a mixture of 1 μL of Live-Dye and 1 μL of PI in 1 mL of Staining Buffer. The cell-laden construct with staining solution was incubated at 37 °C for 15 min, and then solutions were removed. The construct was washed three times with sterilized PBS and observed under an Olympus FluoView™ FV1000 laser scanning confocal microscope (Olympus NDT Inc., Boston, MA, USA) using the red and green fluorescence filters on days 1, 3, and 7. Three slides were used for the confocal microscope. ImageJ was used for the automated counting of red- and green-stained HUVECs in z-axis projections, and viable cell percentage was calculated.

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