Substrates for Static Experiments

For the static experiments, we produced comparatively large gels to increase the probability of finding a spot where the platelets attach while the substrate re-mained as flat as possible at a certain height. We used two different glass slides for the polymerisation, a 24-by-24 mm square glass slide on which we bound the gel and a 18 mm round cover slip to shape the gel.

To start, all glass slides were cleaned with iso-propanol and dried with nitrogen gas. For all following steps, the cover slips were kept in cleaned petri-dishes for convenience. Furthermore, henceforth, all work was done underneath the fume hood for safety reasons as several of the following substances emitted toxic fumes.

To facilitate that the gel was able to bind covalently to the glass slide, the sur-face was pre-coated. The chemical reactions can be found in Fig. 4.2. To start,

Fabrication of PAA Gels 4.2

Table 4.1.: Composition of various buers and solutions used during all experiments.

Buffer / Solution Chemical Composition

APS working solution 10 mg APS in 100µL MilliQ water

Fibrinogen 100 mg in 5 mL MilliQ water

Fibrinogen, Alexa 488 labelled 5 mg dissolved in 3.33 mL 0.1 M sodium bicarbonate, pH 8.3

fluorescent beads stock solution, 40 nm diameter, 505/515 nm excita-tion/emission wavelength

10 µL fluorescent beads, 5 % solids, in 240µL MilliQ water

fluorescent beads stock solution, 100 nm diameter, 660/680 nm excita-tion/emission wavelength

10 µL fluorescent beads, 2 % solids, in 90 µL MilliQ water

fluorescent beads stock solution, 200 nm diameter, 660/680 nm excita-tion/emission wavelength

10 µL fluorescent beads, 2 % solids, in 90 µL MilliQ water

Hepes buffer, 0.5 M, pH 8.0 119.15 g Hepes in 1 L sterile distilled water; working solution 50 mM Hepes-Tyrode buffer, pH 7.4 134 mM NaCl, 12 mM NaHCO3,

2.9 mM KCl, 1 mM MgCl₂, 5 mM HEPES, 5 mM glucose, 0.34 mM Na2HPO4

PBS, 10X, pH 7.2 1.37 M NaCl, 27 mM KCl, 43 mM

Na₂HPO₄*12H₂O, 14 mM KH₂PO₄; working solution 1X

PBS-Glutaraldehyde, 0.5 % 357 µL 70 % glutaraldehyde in 50 mL 1X PBS

Pipes Saline Glucose (PSG), pH 6.8 5 mM PIPES, 145 mM NaCl, 4 mM KCl, 1 mM MgCl2*6H2O, 5 mM glu-cose, 0.05 mM Na₂HPO₄

Prostaglandin E₁ (PGE₁) 1 mg PGE₁ in 940µL DMSO

Sulfo-SANPAH 50 mg in 250 ml of 50 mM Hepes

buffer

Thrombin 1 mg thrombin (1kU/mL) in 2.5 mL

BSA stock solution

Chapter 4 MATERIALS AND METHODS

Table 4.2.: Chemical composition of 10 mL PAA pre-mixed solution. Remaining volume is lled up with 1X PBS. The elastic modulus was measured by rheology by A. Paknikar.

Elastic Modulus [kPa] 40 % Acrylamide [mL] 2 % Bis-Acrylamide [mL]

19.4±0.5 2 0.7

29.3±0.5 2.5 0.75

41.2±_{0.9 2.5 1.3}

54.1±0.7 2.5 2.25

83.1±0.3 3.75 1.5

the glass was wetted with 0.1 M NaOH using a cotton swap and left to dry.

Subsequently, APTMS (3-aminopropyltrimethoxysilane) was applied, again with a cotton swap, and incubated for 5 min (panel A). Excess APTMS was washed away with MilliQ (purified) water until no white residual crystals were visible anymore. Larger remaining water droplets were removed with a low-lint wipe without touching the glass slide itself while the residual water was air dried. Due to the reaction of the silicon oxide in the glass and the silane, a Si-O-Si-bound was established on the glass surface, exposing a free amine group. Subsequently, 0.5 % PBS-glutaraldehyde (compare Table 4.1) was pipetted onto the glass slides and incubated for 30 min (panelB). The supernatant was aspired, discarded sepa-rately and residual solution washed away with MilliQ water. The glutaraldehyde polymerised, leaving a carbon-hydroxide group of the glutaraldehyde free to react with the acrylamide (panelC). The coated side of the glass was then marked and air dried before usage.

In parallel, the round cover slips were treated to be hydrophobic as to avoid any sticking of the gel to the glass. Here, the glass slides were wetted on both sides with Plus One Repel Silane (compare Table 4.3) and incubated for 5 min. They were then washed with 70 % ethanol followed by MilliQ water and air dried until usage.

Subsequently, all solutions needed for the PAA gel were prepared. First, the acrylamide/bis-acrylamide solutions were mixed according to Table 4.2. The cat-alyst APS (ammonium persulfate) was prepared from powder and kept in the fridge until usage. The stock solution of the 40 nm diameter fluorescent beads (Table 4.1) was homogenised by inserting the bead containing tube into the ultra-sonic bath for 10 min followed by vortexing. 485 µL pre-mixed PAA solution of the desired stiffness was mixed with 15µL fluorescent beads.

The next step was the polymerisation of the PAA substrates. As the

polymeri-Fabrication of PAA Gels 4.2

Figure 4.2.: A sketch of the binding of the PAA substrate to the glass slide. A On the glass surface, free Si-O groups bind to the APTMS by building Si-O-Si bonds. B The glutaraldehyde further covalently binds to the NH group and polymerises. C Finally, the acrylamide binds to the CH group of the glutaraldehyde, facilitating the covalent attachment of the PAA substrate to the glass slide. The gure is based on Ref. [107].

Chapter 4 MATERIALS AND METHODS

A B

Figure 4.3.: A sketch of the assembly of the PAA substrate for static experiments. A On the circular coverslip, the acrylamide-bisacrylamide solution (brown) containing beads (green) is pipetted and gently attened with the larger, rectangular glass slide. B The substrate is left to polymerise in an upside-down position on a at surface (black).

sation reaction was initiated as soon as the catalysts are added to the solution, it was essential to work fast to reach a good gel quality. 0.5 µL TEMED (Tetram-ethylethylenediamine) and 5µL APS were added into the PAA-bead-mixture and the solution mixed by pipetting up and down in between each addition. Fol-lowing, 7.5 µL of the final PAA solution was pipetted onto each circular cover slip. The square glass slide was carefully added onto the mixture with the coated surface facing towards the solution thus building a sandwich-like construction of glass-solution-glass (compare Fig. 4.3). The gel was polymerised for 1 h on a flat surface upside-down to ensure that as many beads as possible were found on the upper part of the finished gel. To avoid photo-bleaching of the beads during polymerisation, everything was covered with aluminium foil.

After polymerisation, the glass sandwiches were immersed into 1X PBS inside a petri-dish with the correct orientation, the circular glass slide facing up. The upper glass started to detach and was then be removed carefully with a pair of tweezers. The gels were then washed in PBS to remove residual, unpolymerised PAA solution. From this point onwards, the gels were kept moist at all times.

Lastly, the gels were coated with a layer of fibrinogen to facilitate the attachment of the platelets. This was done by first binding a cross-linker, Sulfo-SANPAH (Ta-ble 4.3), onto the substrate. As Sulfo-SANPAH is a cross-linker activated by UV light and light-sensitive, the following was done with as low room illumination as possible, avoiding direct light completely. The substrates were covered with Sulfo-SANPAH and incubated under UV light, wavelength 366 nm, for 8 min.

Afterwards, the gels were washed in Hepes buffer. This procedure was then re-peated once more before the fibrinogen was added onto the substrates. For all static experiments, unlabelled fibrinogen was used. Here, 5 µL fibrinogen was

Fabrication of PAA Gels 4.2 mixed in 995µL 1X PBS to a final concentration of 0.1 mg/mL and added to gels

to cover them entirely. The substrates were incubated at 4^◦ C over night. The following day, the gels were washed again in PBS and stored in PBS until usage.

The finished gels were used for one week after which they started to loose their flat appearance.