3.2.1 Casting of Polydimethylsiloxane (PDMS) Substrates
For the studies presented here, an inverted research microscope equipped with objectives with a working distance between 200 and 300µm (see section 3.6 for de-tails) was used. As a consequence, it was necessary to use a substrate thin enough to allow for focusing on the top plane of the substrate in the case of live imaging of platelet spreading. Furthermore, a thin substrate also facilitated the mounting of fixed samples.
To obtain such thin substrates, a mixture of 10 parts PDMS-elastomer (Sylgard® 184 Silicone Elastomer Base, Dow Corning, Midland, Michigan, USA) and 1 part of the corresponding cross-linker (Sylgard® Silicone Elastomer Curing Agent, Dow Corning) was spin coated onto the structured silicon wafer (see figure 3.2 a)).
The spin coating was carried out for 2 minutes at a speed of 1000 rpm with a ramp time of 2 seconds (Spincoat G3P-8, Specialty Coating Systems (SCS), Surrey,
Chapter 3 MATERIALS AND METHODS
Figure 3.2.: Sketch of PDMS-substrate casting.
a) A base/cross-linker-mixture for PDMS was spin coated onto the structured wafer and cured afterwards for1hour at80◦C on a hotplate.
b) The desired structure was cut from the PDMS cast and transferred with the structured side facing up onto a cover slip.
United Kingdom). Afterwards, wafer and PDMS were baked for 1 hour at 80◦C on a hotplate. After being cured, the desired structure was cut out and transferred (structured site to the top) onto a cover slip (see figure 3.2 b)). For the exper-iments with fixed, actin-stained platelets, No.1 (24×60 mm, VWR, Darmstadt, Germany) cover slips were used, whereas for the experiments with non-fixed, membrane-stained platelets, No.1 round cover slips (∅ 50 mm, VWR) were used due to different requirements during imaging.
3.2.2 Substrate Coating with Fibrinogen
Completely Coated Substrates
Complete fibrinogen coating of substrates, produced as described in section 3.2.1, was accomplished by activation in a plasma-cleaner (Plasma Cleaning, PDC-32 G, Harrick Plasma, Ithaca, New York, USA) at level “medium” for 1.5 minutes. The plasma-cleaning renders the substrate hydrophilic and therefore allows for com-plete coating. Afterwards, a square of approximate size of 18×18 mm was drawn around the substrate (onto the cover slip) with a mini PAP pen (Invitrogen, Darm-stadt, Germany). The drawn boundary prevented liquid from running down the substrate and thus kept the later applied fibrinogen solution in place. 500 µl of Alexa Fluor®488 conjugated fibrinogen solution (concentration of 0.05mgml, fibrino-gen from human plasma, Alexa Fluor® 488 conjugate, excitation approximately 495 nm, emission approximately 519 nm, Invitrogen) were added to the activated substrate and incubated for 1 hour at room temperature in dark (see figure 3.3 a)).
Afterwards, the fibrinogen solution was removed and the substrate was washed
26
Substrate Production 3.2
Figure 3.3.: Sketch of dierent steps in complete substrate coating.
a) The substrate was activated in a plasma-cleaner and thus rendered hydrophilic. This proce-dure allowed complete coverage of the substrate with brinogen by incubation for1 hour with brinogen solution. A 18×18 mm square drawn with a mini PAP pen around the substrate prevented the uid from running down the substrate.
b) The substrate was washed three times with PBS and either let dry or kept in PBS.
three times with phosphate buffered saline (PBS)i. A sketch of the resulting coated substrate is shown in figure 3.3 b). The substrate was either stored in PBS or left to dry.ii Dry storage only took place for some of the substrates used to obtain the data presented in chapter 5. Usually, the substrates were prepared one day prior to the experiment and stored at 4−8◦C until usage.
Selectively Coated Substrates
Selective coating of PDMS-substrates was here achieved by microcontact printing (MCP) [82]. The method explained below has been adapted from the method described by Lianget al. in [60].
A sketch of the different steps in selective coating of the substrates is shown in figure 3.4. Transfer of protein onto the substrate was achieved by a stamp made of PDMS. This stamp was produced by mixing 10 parts PDMS-elastomer (Sylgard® 184 Silicone Elastomer Base, Dow Corning) and 1 part of the corresponding cross-linker (Sylgard®Silicone Elastomer Curing Agent, Dow Corning) and pouring the mixture into a petri-dish. Then the mixture was degassed in a desiccator before being baked in an oven (DryLine, VWR) for 1.5 hours at 65◦C. Afterwards, a piece was cut from the PDMS-layer and coated with Alexa Fluor® 488 conjugated fibrinogen solution (concentration of 0.05mgml, Invitrogen) by incubating the stamp for 1 hour with the solution at room temperature in dark (see figure 3.4 a)). Then, the solution was removed from the stamp, which subsequently was washed twice,
i0.137 M NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4·12 H2O, 1.4 mM KH2PO4
iiThe differences in substrate coating resulting from wet or dry storage are shown in figure 3.5.
Chapter 3 MATERIALS AND METHODS
each time for about 5 seconds in fresh MilliQ water and the remaining liquid was blown off with dry nitrogen as described in [74].
A structured substrate, produced as described above (see section 3.2.1), was ac-tivated using plasma-cleaning (Plasma Cleaning, PDC-32 G, Harrick Plasma) at level “medium” for 1.5 minutes. The dried stamp was then placed and briefly pressed onto the activated substrate so that the side of the stamp coated with fib-rinogen touched the structured side of the substrate (see figure 3.4 b)). The stamp was kept on the substrate for 15 minutes, during which a weight of about 15 g was placed on top of the stamp. The stamp and the weight were taken off and the selectively coated substrate was stored at 4−8◦C.iii To prevent the liquid running down the substrate, a square of the approximate size of 18×18 mm was drawn around the substrate (onto the cover slip) with a mini PAP pen (Invitrogen) (see
iiiThe substrates were usually produced one day prior to the experiments.
Figure 3.4.: Sketch of dierent steps in selective substrate coating.
a) A PDMS stamp was coated by incubation with brinogen solution for1 hour. Afterwards it was washed twice in MilliQ water and dried with dry nitrogen.
b) Activation of the substrate in a plasma-cleaner rendered it hydrophilic and allowed for protein transfer when the stamp was placed onto the substrate.
c) The stamp was removed and a18×18 mm square was drawn around the substrate with a mini PAP pen in order to keep liquid from running down the substrate.
d) Poloxamer407was added to the substrate to block unspecic binding sites and incubated for 2 hours. Thereafter, the substrate was washed again three times with PBS.
28
Characterization of Substrates 3.3 figure 3.4 c)).
Unspecific binding sites were blocked by incubation of the substrate with 0.2%
Poloxamer 407iv(Sigma-Aldrich, St. Louis, Missouri, USA) for 2 hours on the day of the experiment. Afterwards, the liquid was removed and the substrate was washed three times with PBS. The resulting coating is sketched in figure 3.4 d).