Biology Reference
In-Depth Information
6.
Return to
step 2
to continue stamping more substrates. After all substrates are
stamped, proceed to
step 7
.
7.
Using a Pasteur pipet, deliver a solution of EGT dropwise onto each substrate
until the liquid covers it entirely. This usually requires approx 0.5-1 mL per
square inch of substrate. Incubate with EGT for 30 min.
8.
Using forceps cleaned with ethanol and blown dry, grasp the corner of the sub-
strate and rinse with a stream of ethanol on both sides of the pattern for 20 s.
Place the substrate on a clean surface, and rinse the forceps with ethanol. Grasp
the substrate again in a different location and rinse again with ethanol to wash the
area previously masked by the forceps.
9.
Blow the ethanol off the substrate with pressurized air or nitrogen.
10.
The stamped substrates should be placed into containers taking care not to allow
the patterned surface to rub against coarse surfaces. They are stored under nitro-
gen gas in a cool, dark location. Place the containers in ziplock bags that are
filled with nitrogen.
3.2. Coating Stamped Substrates
with ECM Proteins and Plating Cells
1.
To coat the substrates with ECM, make a solution of the protein (25
g/mL) in
PBS. Typically, 0.25-mL solution per square inch of substrate is sufficient.
µ
2.
Place a 0.25-mL drop of ECM solution onto bacteriological Petri dishes or
another disposable hydrophobic surface. Float each substrate, with patterned side
face down, onto the drops. Let sit 2 h at room temperature.
3.
After 2 h, add a large amount (5-15 mL) of 1% bovine serum albumin (BSA,
Fraction V, Sigma, St. Louis, MD) dissolved in PBS directly to dish. Remove
substrates and place directly into plating medium (remember to flip slide so pat-
tern is facing up again).
4.
Plate cells directly on substrates using standard experimental technique.
4. Notes
1.
Gold Slide: Choice of glass: We find No. 2 glass cover slips to be less likely to
break than No. 1, although at the same time not too heavy to pick up with forceps.
We have successfully used standard histology mounting slides as well.
2.
Gold Slides: Evaporation: We recommend using an evaporator rather than a sput-
ter coating system to coat the substrates for several reasons. Most sputterers are
single source, and are impractical for coating two different metals (Ti and Au) on
a substrate. Sputtering also gives less homogeneous films that would require an
additional annealing step to correct. And last, sputtering systems generally pro-
duce films with higher quantities of metal oxides and other impurities that would
interfere with the generatin of the SAM surface.
3.
Gold Slides: Storage: Typically, gold-coated substrates become “mottled” after
4-5 wk and are no longer deemed suitable for experiments; streaks with heterog-
enous transparency develop (they are obvious to the naked eye). This may be
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