PDMS cleansing. Prior to use, all newly fabricated PDMS microfluidic devices were Soxhlet
extracted with ethyl acetate for 6 hours.54 After a single use, the stamps were immersed in CH2Cl2 overnight to extract leftover thiols from the PDMS, and then placed in a vacuum chamber for 2 hours to remove the volatile solvent from the PDMS.
SPREAD Inking and Printing Procedure. The cleaned PDMS cassettes were sealed onto the
surface of a glass microscope slide, then 5mM hydroxylamine-undecane thiol in 1:1 EtOH/CH2Cl2 was flowed into the microfluidic device and allowed for 30 seconds to permeate the PDMS. The device was then evacuated with the aid of negative pressure, and the thiol was given time to diffuse into the PDMS (12 minutes for microarray stamp inking, 4 minutes for µCP inking). After diffusion, the PDMS stamp was removed from the glass
surface, and pressed onto a gold coated microscope slide for 15s. After printing, the surfaces were backfilled with EG4 to create surface inert to cell attachment.
Fluorescence Visualization. For visualization, the surfaces were exposed to a fluorescent
ketone functionalized rhodamine12 (10 mM, 3 hours). After dye immobilization, a thin piece of transparent scotch tape was placed on the surface, the SAM was thermally desorbed at 80
◦
C for 20 min, allowed to cool to RT, then carefully removed and attached to a clean glass substrate.
RGD-ketone Functionalization. Surfaces were exposed to a 10 mM solution of RGD-ketone
for 3 hours, and then rinsed thoroughly with water.
Cell Seeding- For single cell assays, cells were trypsinized and then diluted to a
concentration of 30,000 cells/mL. Cells were seeded onto the surfaces in DMEM solution only for 2 h. For confluent tissue assays cells were seeded at a density of 500,000 cells/mL for 3 h under otherwise identical conditions. Cell polarity was determined by immunostaining and fluorescence microscopy. To determine cell division vectors, cell nuclei were observed and a line was drawn between the center of the nuclei. This represented the vector of cell division.
Immunostaining for Fluorescence Microscopy. Adherent cells were fixed with 3.2%
paraformaldehyde in PBS for 10 min and then permeabilized with 0.1% Triton-X100 in PBS (PBST) for 10 min. For polarity, cells were stained with mouse anti-gigantin antibodies (1:400, BD biosciences) and phalloidin-tetramethylrhodamine B isothiocyanate (1:250, Sigma) in PBST containing 5% goat serum for 1 h, followed by FITC-conjugated goat anti- mouse IgG (1:400 in PBST, Jackson ImmunoResearch), phalloidin-tetramethylrhodamine B isothiocyanate (1:50 in PBST, Sigma), and DAPI (4’, 6-diamidino-2-phenylindole
62
dihydrochloride, Sigma) (1:500 in PBST) in 5% goat serum/PBST for 1 h. Substrates were rinsed with deionized water before being mounted onto glass cover slips for microscopy. All optical and fluorescent micrographs were imaged using a Nikon inverted microscope (model TE2000–E). All images were captured and processed by MetaMorph.
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