Electronic Supplementary Material (ESI) for Analyst.
This journal is © The Royal Society of Chemistry 2016
Electronic Supplementary Information for
Oxygen as a chemoattractant: confirming cellular
hypoxia in paper-based invasion assays
Andrew S. Truonga and Matthew R. Locketta,b*
a Department
of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, 125
South Road, Chapel Hill, NC, 27599-3290
b
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 450 West Drive,
Chapel Hill, NC 27599-7295
* Author to whom correspondence should be addressed: [email protected]
Figure S1: Representative schematics depicting the different culture configurations used in this work. The
green-black lines represent the cell-seeded layers. The pink-black lines represent the layers with only Matrigel.
The blue lines represent the cellulose acetate films: broken lines = perforated films, solid lines = non-perforated
films. Note: schematics are not drawn to scale.
Figure S2: Calibration curves of fluorescein diacetate stained MDA-MB-231 cells. Known numbers of cells
were suspended in Matrigel, seeded into the hydrophilic zones of the paper-based scaffold, and incubated for
four hours before being washed twice with 1X PBS and then stained with fluorescein diacetate (10 µM for 10
min at 37 °C). Each sheet was washed again twice with 1X PBS before being imaged on a Typhoon 9400
scanner (GE Life Sciences) at a resolution of 200 µm; the fluorescence intensity of each zone was obtained
using ImageJ. The plotted fluorescence values are background subtracted to account for the background
fluorescence of the paper-based scaffolds. Each data point represents the average and standard deviation of 30
replicate zones. The black line in the graph represents the least squares sigmoidal four-parameter logistic curve
(R2 = 0.960).
Figure S3: Calibration curves of eGFP-expressing MDA-MB-231 cells. Known numbers of (left) M231-eGFP
and (right) M231-HREeGFP cells were suspended in Matrigel, seeded into the hydrophilic zones of the paperbased scaffolds, and incubated for four hours before imaging with a Typhoon 9400 scanner (GE Life Sciences)
at a resolution of 200 µm. The fluorescence intensity of each zone was obtained using ImageJ. The plotted
fluorescence values are background subtracted to account for the background fluorescence of the paper-based
scaffolds. Each data point represents the average and standard deviation of five replicate zones. The black line
in each graph represents the least squares linear regression for the M231-eGFP (R2 = 0.985) and M231HREeGFP (R2 = 0.991) cell lines.
Figure S4: Average eGFP fluorescence intensity of 47,000 M231-mCherry cells with a hypoxia-responsive
enhanced green fluorescent protein gene in the presence and absence of Whatman Nuclepore polycarbonate
track-etched (TE) membranes after 24 h of incubation. The track-etched membranes were used to sequester the
cells in particular locations of the invasion stacks. In both setups, the cells were seeded and placed in the
proximal position of an invasion assay in the closed configuration. In one stack (TE), the cell-containing layer
was sandwiched between two track-etched membranes. In another stack (no TE), only one track-etched
membrane was used to prevent the cells from invading apposed layers; there was no track-etched membrane
between the cell-containing layer and the fresh medium. Each sheet was imaged for both eGFP and mCherry on
a Typhoon 9400 scanner (GE Life Sciences) at a resolution of 200 µm; the fluorescence intensity of each zone
was obtained using ImageJ. Each bar represents the average and standard deviation of five replicate zones. The
levels of eGFP expressed per cell in the two stacks were statistically indistinguishable (p = 0.134).
Figure S5: Fluorescence intensity of fixed numbers of M231-mCherry cells with a hypoxia-responsive
enhanced green fluorescent protein gene in the absence (control) and presence (induced) of 200 µM CoCl2.
Induced cells were treated with CoCl2 for 24 h before analysis, to induce the transcriptional activity of HIF-α
proteins. In both setups, known numbers of cells were resuspended in 100µL of 1X PBS, seeded in a white 96well plate, and immediately lysed with 50µL of Mammalian Protein Extraction Reagent (Pierce Biotechnology).
The mCherry fluorescence intensity of each sample was measured with SpectraMax M5 Microplate Reader
(Molecular Devices) and the mCherry fluorescence intensity of the control and induced samples compared.
Each bar represents the average and standard deviation of three technical replicates. These data indicate that the
constitutive expression levels of mCherry are not influenced by the expression of eGFP.
Table S1: Summary tables of the percentage of cells that invaded neighboring layers from the original
intermediate layer (layer 0) after 48 h of incubation. A) The “closed configuration” in which we placed a solid
cellulose acetate film at one end of the invasion stack and a perforated cellulose acetate film at the other to
generate a monotonic gradient of oxygen, soluble nutrients, and cellular waste products; the perforated film
allowed the free exchange of the stack with the culture medium. B) The “open configuration” in which both
ends of the invasion stack contained a perforated cellulose acetate film. C) The “decoupled configuration” in
which we placed a 100-µm slab of PDMS at one end of the invasion stack and a perforated cellulose acetate
film at the other to generate a monotonic gradient of soluble nutrients and cellular waste products, but allow the
free exchange of oxygen on both ends of the culture stack.
Table S2: Summary tables of the percentage of cells that invaded neighboring layers in the closed and open
configurations from the A) distal, and B) proximal locations after 48 h of incubation. N.D. = not detected.