In instances were it is stated that all liquid is removed, this does not include the thin liquid film that remains each time after removal of liquid.
3.5.1 Actin Staining
Platelets were diluted to a concentration of 6×107 cellsml in Medium 199 (Bio-Whit-taker® Medium 199, with Earle’s Balanced Salt Solution, with L-Glutamine and HEPES, Lonza). The substrates (prepared as described in section 3.2.2) were washed three times with Medium 199 (Bio-Whittaker® Medium 199, with Earle’s Balanced Salt Solution, with L-Glutamine and HEPES, Lonza) and stored with added Medium 199 (Bio-Whittaker® Medium 199, with Earle’s Balanced Salt So-lution, with L-Glutamine and HEPES, Lonza) until usage. Prior to the addition of Figure 3.8.: Description of platelet counting with a micro-hematocrit capillary.
By reading out the height of the liquid column and the height of the pellet, the num-ber of platelets can be deter-mined with the help of a ZIP-ocrit, Microhematocrit Cen-trifuge Reader Card.
34
Platelet Staining and Sample Preparation 3.5 platelets all liquid was removed. The sketch in figure 3.9 shows the different steps
of sample preparation in the case of fixed, actin-stained platelets.
150µl of diluted platelet solution (prepared as described above, section 3.4) were added to each substrate and given 15 minutes to sediment onto the substratesviii (see figure 3.9 a)) before addition of 50 µl of thrombin solution (4 mlu, thrombin from human plasma, ≥ 2800 NIH units/mg protein, Sigma-Aldrich). The sub-strates together with the platelet/thrombin-mixture were placed into an incubator (HeraCell 150, Thermo Scientific) at 37◦C and 5 % CO2 for 60 minutes in order to let the platelets spread (see figure 3.9 b)). The platelets were fixed by adding
viiiThis yields a larger number of spread platelets.
Figure 3.9.: Sketch of dierent steps in preparing the xed, actin-stained platelet samples.
a)150µl of diluted platelet solution were added to a washed substrate.
b) Then,50 µl thrombin solution were added after 15minutes and the sample was incubated for60minutes in an incubator at 37◦C and5%CO2.
c) 200 µl of formaldehyde solution were added to x platelets and the sample was incubated again for20minutes at37◦C and5%CO2and then washed three times with PBS.
d) Afterwards,200µl of Alexa Fluor® 594phalloidin were added and incubated for30minutes at room temperature under the exclusion of light and subsequently washed again three times with PBS.
e) Most liquid was removed and the sample was sealed by addition of ProLong® Gold Antifade reagent and a cover slip, which was then attached to the underlying cover slip by nail polish.
Chapter 3 MATERIALS AND METHODS
200µl of approximately 4 % formaldehyde (FA) solution (∼4−8◦C, diluted from min. 37 % formaldehyde (with 10 % methanol for stabilization), Merck KGaA, Darmstadt, Germany) to the platelet/thrombin mixture and incubating it again for 20 minutes at 37◦C and 5% CO2 (see figure 3.9 c)). Subsequently, all liquid was removed and the substrates were washed three times with PBS. Again all liq-uid was removed and 200 µl Alexa Fluor® 594 phalloidin (approximately 4.9 mlu, excitation: approximately 590 nm, emission: approximately 617 nm, Invitrogen) were added. The samples were incubated for 30 minutes at room temperature un-der the exclusion of light. Finally, all liquid was removed and the substrates were washed again three times with PBS (see figure 3.9 d)). The cover slip to which the substrate was attached, was transferred to a microscopic glass slide for easier handling and attached to it with nail polish. Most of the remaining liquid was removed from the cover slip and ProLong® Gold Antifade reagent (Invitrogen) was brought onto the substrate. This reagent helps to prevent photobleaching and cures so that long-term storage is possible. In order to finally seal the sample, a 18×18 mm cover slip (No.1, VWR) was put on top of the ProLong®Gold Antifade (Invitrogen) and fixed to the underlying cover slip with nail polish (see figure 3.9 e)). The sample was left for some time at room temperature to start the curing of the ProLong® Gold Antifade (Invitrogen) and then stored at 4−8◦C.
3.5.2 Live Plasma Membrane Staining
Staining platelets with CellMask™dye (Life Technologies GmbH, Darmstadt, Ger-many) has been conducted for example in [52]. The concentrations for BSA and platelets in the following protocol have been taken from this reference. The con-centration of the CellMask™ dye (Life Technologies GmbH) has been chosen as proposed in [64]. A sketch of the different steps in membrane staining is depicted in figure 3.10.
A staining solution was prepared by mixing HT buffer (supplemented with 5mgml BSA) and CellMask™ DeepRed (excitation: approximately 649 nm, emission: ap-proximately 666 nm, Life Technologies GmbH) to yield a final dye concentration of 2.5 µgml. Approximately 5µl of unstained platelet solution were mixed with about 995µl of this staining solution to yield a final platelet count of 2×107 cellsml in a total liquid amount of 1 ml (see figure 3.10 a)). This mixture was incubated for 6 minutesix. At the end of incubation, 25µl of PGE1-solution (∼ 0.106mgml, Cayman
ixabout 4 of the 6 minutes in an incubator (HeraCell 150, Thermo Scientific) at 37◦C and 5% CO2
36
Platelet Staining and Sample Preparation 3.6
Figure 3.10.: Sketch of platelet preparation for live imaging of spreading.
a) Platelets were mixed with a staining solution containing CellMask DeepRed dye and incu-bated for6minutes (∼4 of the6 minutes in an incubator at37◦C and5%CO2).
b) The solution was centrifuged for5minutes at480g and20−21◦C to separate platelets from liquid. Then, the cell pellet was re-diluted in HT buer (supplemented with5mgml BSA).
c)150 µl of stained platelet solution were pipetted onto a washed substrate.
d) Shortly thereafter50 µl of thrombin solution (16 mlu) were added to the platelet solution.
Chemical Company) were added. Afterwards, the mixture was centrifuged for 5 minutes at 480 g and 20−21◦C in a centrifuge (Eppendorf Centrifuge 5810 R, Eppendorf). The resulting cell pellet was resuspended in 1 ml HT buffer (sup-plemented with 5mgml BSA (see figure 3.10 b)). The BSA reduces background fluo-rescence [92]. A structured, completely fibrinogen-coated substrate (prepared as described in section 3.2.2) was washed three times with HT buffer (supplemented with 5mgml BSA). After the last washing step, the liquid was removed and HT buffer (supplemented with 5mgml BSA) was added for storage.
The following steps were performed on a microscope (see section 3.6 for details) equipped with an incubation-chamber (37◦C, 5% CO2, model INUG2E-ONICS, Incubation System for Microscopes, Tokai Hit, Ltd., Gendoji-cho, Fujinomiya-shi, Shizuoka-ken, Japan). A washed substrate was placed into the incubation chamber and nearly all liquid was removed from the substrates. Then, 150µl of membrane-stained platelet solution were added to the substrate (see figure 3.10 c)) and shortly thereafter 50µl of thrombin solution (16mlu, thrombin from human plasma,≥2800 NIH units/mg protein, Sigma-Aldrich) were added to trigger spreading (see figure 3.10 d)). Details of imaging can be found in the next section (section 3.6).
Chapter 3 MATERIALS AND METHODS