Download Trypan blue exclusion was done by resuspending the cells in

Supplemental Materials
Trypan Blue Exclusion and Invitrogen Live/Dead Stain
Trypan blue exclusion was done by resuspending the cells in medium after trypsinizing,
adding 0.1 ml 0.4% solution of trypan blue to 1ml of cells, and counting the number of blue
stained cells (dead) and the total number of cells using a hemocytometer. The percent viable was
then determined.
The Invitrogen Live/Dead Stain was prepared by adding calcein-AM and ethidium
homodimer-1 to Dulbecco’s Phosphate Buffered Saline (DPBS) (final concentration of 2µM for
each fluorescent dye). The medium was removed from the cells, the cells were washed once
with DPBS, stained for 45 min at 5% CO2 and 37oC and observed under a fluorescent
microscope using FITC filter (494nm excitation/517nm emission) and RFP filters (528nm
excitation/617nm emission). The Live/Dead Stain discriminated live from dead cells
simultaneously because the green-fluorescent calcein-AM indicates intracellular esterase activity
(live) and red-fluorescent ethidium homodimer-1 indicates loss of plasma membrane integrity
(dead). Micrographs were assessed using ImageJ to determine viability based on areas of cells
that imbibed the fluorescent dyes.
Albumin Assay
We coated the wells of a 96-well plate with goat anti-human albumin antibody, washed
the wells with buffer and blocked with blocking buffer in the refrigerator overnight. We then
transferred 100 µl of the medium samples (diluted 1:50 in sample diluent) into the coated wells.
After 1 hour incubation at room temperature, we added HRP-conjugated goat anti-human
antibody (1:75,000 dilution) to the wells and incubated again for 1 hour. Following washing
steps with buffer, we added 100 µL of enzyme substrate (tetramethylbenzidine) and incubated in
the dark for 15 min. After adding stopping solution, we measured the absorbance of the solution
using a plate reader at 450 nm.
P450 Assay
The induction reagents (25 M of rifampicin for CYP3A4 and 1 M of 3-methylcholanthrene for CYP1A1 induction) were diluted in medium, added to each device and each
static control designated for induction for 48 hours. After 48 hours, we washed the induced and
noninduced groups with serum-free device medium (as described in by Promega) five times for 5
minutes each. Then we added Luciferin-CEE (the substrate for 1A1) for a 3 hour incubation at
37oC and we removed a medium sample for P450 1A1. The devices and controls were washed
again, Luciferin-IPA (substrate for 3A4) was added, incubated for 1 hour at 37oC and then we
removed a medium sample for P450 3A4. The collected medium was frozen at -80oC until the
assay was run. We transferred 50 µl of the samples into the wells of a white opaque 96 wellplate (Corning #3912). Then 50 µl of the detection reagent specific for the desired enzyme was
added to the appropriate wells, incubated for 20 min at room temperature and luminescence was
read with a Veritas luminometer using the settings provided by the manufacturer.