Bmg omega

The PicoGreen assay (Invitrogen) used as a measure of viable cells by detecting total double stranded DNA, was performed according to the manufacturer's instructions. Briefly, DNA was extracted with 0.5 mg/mL proteinase K (Roche) phosphate buffered EDTA (PBE). Samples were diluted (1∶10) in RNaseA solution (Invitrogen) and incubated with PicoGreen assay reagent before being read on a fluorescence plate reader (BMG Omega, BMG LABTECH; excitation 485 nm emission 520 nm).

To establish cell metabolism over 14 days of microaggregate growth, the Alamar blue (Invitrogen) assay was used as per the manufacturer's instructions. On days 1, 3, 7 and 14 a 3% volume of Alamar blue was added per well. The plates were incubated at 37°C for 1 hr and 100 µl medium transferred to a 96-well black plate. Fluorescence (excitation 544 nm, emission 590 nm) was detected using a plate reader (BMG Omega, BMG LABTECH). WST-1 (Roche) was used to quantify the viability of RWPE-1 and RWPE-2 cells cultured in different medium types. Cells were cultured for 3 days followed by the addition of 10% WST-1 for 1 hr before reading absorbance at 450 nm on a Bio-Rad Benchmark Plus plate reader.