Prestoblue pb reagent

SF films were sterilised by immersion in 70% (vol.) ethanol during 10 minutes and washed twice with sterile 1X PBS dissolution before the seeding. The cells were detached using 0.05% trypsin/EDTA and seeded on the films at a density of at 5∙10³ cells∙cm⁻² with 1 mL of DMEM/F-12 (1:1) expansion medium. Tissue culture polystyrene substrates (TCPS) were also seeded to be used as positive controls. Cell proliferation was evaluated 2 d, 4 d, 7 d and 10 d after seeding, using PrestoBlue (PB) reagent (Invitrogen, Thermo Fisher Scientific,Waltham, MA, USA). This is resazurin-based membrane permeable solution which does not require cell lysis. PB quantitatively analyses proliferation of metabolically active cells by mitochondrial reduction of resazurin to a red fluorescent compound called resorufin. As a consequence, the reagent exhibits a change in colour, as well as a shift in its fluorescence. Following the manufacturer’s protocol, a 10% solution of PB was added to the wells and incubated for 4 hours at 37 °C in a 5% CO₂ humidified atmosphere. The solution was then removed and relative fluorescence (RF) was measured using a Synergy MX microplate reader (Biotek Instruments, VT, USA) with an excitation wavelength of 570 nm and an emission wavelength of 610 nm.

Cell viability was measured using PrestoBlue (PB) reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Briefly, a mixture of 10% PB was added to cells in the culture media. The absorbance was read at 570 nm after RPE cells were incubated for 2 h with PB reagent. Each sample was measured three times. Each experiment was repeated at least three times and two or three samples were obtained from each independent experiment.

PEGDA (Mw 700 Da), LAP, tartrazine, bromocresol green, α-naphtholphthalein powder, glucose, horseradish peroxidase, glucose oxidase, potassium iodide, trehalose, sodium citrate, and Dowex 1 × 4 chloride foam beads were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco (Grand Island, NE, USA). Presto blue (PB) reagent was purchased from Invitrogen (A13261, Waltham, MA, USA). sodium citrate was purchased from VWR, Radnor, PA, USA.