Alamar blue cell proliferation assay

Viability of coated MIN-6 cells using different coating deposition sequence was evaluated using a live/dead staining kit. Briefly on the day of experimentation, 1 μL of calcein acetoxymethylester (Calcein AM (662 Da), indicator for live cells, Invitrogen, USA) and 5 μL of propidium iodide (PI (668 Da), indicator for dead cells, Invitrogen, UK) were added to a 200 μL suspension of coated spheroids in cell culture medium. Cells were incubated in the dark at 37 °C for 30 min. Stained cells were then visualised using confocal laser scanning microscopy (Olympus FV1000, Multiple Ar laser, Germany), and images were analysed using ImageJ software (NIH, USA).
The metabolic activities of coated and non-coated (control) MIN-6 cells were assessed using the Alamar Blue cell proliferation assay (Thermo Fisher, UK). The assay reagent reduces from a non-fluorescent blue colour to a fluorescent red form, during cellular metabolism. Hence, the amount of assay reagent reduction is proportional to cell number (presuming equal metabolic activity). Briefly, 20 μL of Alamar Blue reagent were added to 200 μL of cell spheroids suspension in DMEM, followed by incubating for 4 h at 37 °C. The fluorescence was then read (590 nm emission and 560 nm excitation) using a microplate reader (GloMax-Multi+, Promega, USA). The results were reported as percentage reduction between the coated and the control sample.

The AlamarBlue cell proliferation assay (Thermo Fisher Scientific, USA) has been used to measure the metabolically active cells. The principle behind this assay relates to the fact that metabolically active cells create a reducing environment that promotes the conversion of resazurin (non-fluorescent) to resorufin (highly fluorescent). The absorbance was measured using an ELISA reader/96-well multiscanner (Tecan, Crailsheim, Germany) at ex550/em630 nm.

Hen eggs were obtained from a local supermarket. PCL (Mw = 65000 Da, Sigma Aldrich) was used to produce nanofiber mats. Rabbit anti-ovalbumin antibody was purchased from Merck Millipore (Germany). Alamar Blue cell proliferation assay and Live/Dead kit were acquired from Thermo Fischer (USA). All other chemicals were obtained from Sigma-Aldrich (Germany) and used as received unless otherwise is specified.