Cytation3 automated microscope plate reader

Culture media was collected after every change (n=3), diluted 1:10 in DPBS, and quantified according to manufacturer specifications using the human Albumin ELISA kit (Abcam). Absorbance was read on a Cytation3 Automated Microscope Plate Reader (BioTek). The Human Albumin ELISA has cross-reactivity with bovine albumin, and the albumin content of DMEM + 10% FBS was subtracted from all quantities. Cumulative albumin secretion was determined by summing all quantities and normalizing to cell number which was determined via dsDNA quantification described above.

Activities of cytochrome P450 oxidase (CYP) subtypes 3A4 and 2C9 were quantified using Luciferin-IPA or Luciferin-H substrates, respectively (Promega). Scaffolds (n=3 per enzyme test) were incubated in enzyme substrate for 60 minutes (Luciferin-IPA) or 4 hours (Luciferin-H). Samples were subjected to the lytic protocol, wherein samples (as opposed to supernatant) are incubated with the Luciferin detection reagent. Lysates were assayed for luminescence on Cytation3 Automated Microscope Plate Reader (BioTek). All samples were first normalized to cell number, then to Day 0 activity of tissue culture plastic cultured cells.

Scaffolds were collected 1, 3, 7, 10, and 14 (n=3) days after seeding, 2D controls were trypsinized and frozen (−80°C) until analysis. Scaffolds (cell pellets for 2D) were digested using a solution of 1 μg/mL proteinase K (Sigma) in a 60°C for 24 hours. Digests were assayed using PicoGreen dsDNA Quantification Kit (Molecular Probes) according to manufacturer specifications. Fluorescence was read on a Cytation3 Automated Microscope Plate Reader (BioTek). Percent seeding efficiency was determined by dividing total DNA content of day 1 (24 hr) samples by the DNA content of an aliquot of 0.1×10⁶ cells. Cells per scaffold were determined by normalizing DNA content per scaffold to DNA content per cell.