Alamarblue reagent

Ten thousand cells were seeded in 100 μl of media in each well of a 96-well flat-bottom transparent plate. One-tenth of the volume of AlamarBlue reagent (G-Biosciences, 786–921) was directly added to the wells and incubated for 4 hr at 37°C in a cell culture incubator, shielded from direct light. Results were recorded by measuring fluorescence using a fluorescence excitation wavelength with a peak excitation of 570 nm and a peak emission of 585 nm on a microplate reader (Tecan Infinite F500, Tecan Group Ltd.).

To determine cell proliferation and viability in the 3D scaffolds, Live–Dead fluorescence microscopy and Alamar blue assay were performed according to the previously published method [28 , 29 (link)]. For Live–Dead fluorescence microscopy, 5 mg/mL fluorescein diacetate (FDA) and 2 mg/mL propidium iodide (PI) were used to detect the live and dead cells, respectively. The stained HUVECs were observed under the fluorescent microscope (CKX53, Olympus, Japan). For Alamar blue assay, the culture medium was aspirated after 72 h and the cell‐impregnated constructs were washed with sterile PBS (pH 7.4) and incubated with a freshly prepared Alamar blue reagent (G‐Biosciences, St. Louis, USA) in the complete culture medium (10% v/v) for 4 h. The fluorescence intensity was read by a fluorescence plate reader (BMG LABTECH, POLAR star Omega, German) at the excitation λ_max of 544 nm and emission λ_max of 590 nm. Matrigel was used as the gold standard [30 (link)].