CM acknowledges financial support from the International Patient Organization for C1-Inhibitor Deficiencies (HAEi), Stichting Vrienden van Het UMC Utrecht, and the Landsteiner Foundation for Blood Transfusion Research (LSBR).

The local Medical Ethical Committee of the University Medical Center Utrecht approved the drawing of blood for ex vivo research purposes, including those of this study.
1. Solution Preparation
<ol>
	<li>Prepare a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) Tyrode&#39;s buffer by dissolving 10 mM HEPES, 0.5 mM Na2HPO4, 145 mM NaCl, 5 mM KCl, 1 mM MgSO4, and 5.55 mM D-glucose in distilled water.
	<ol>
		<li>Make two variants of the buffer: set the pH levels at 6.5 and at 7.4, respectively. Make fresh buffer for each day of experiments. Supplement with 10 ng/mL prostacyclin where indicated.</li>
	</ol>
	</li>
	<li>Prepare TRIS-buffered saline (TBS) by dissolving 50 mM Tris and 150 mM NaCl in distilled water and adjust the pH to 9.0.</li>
	<li>Prepare acid citrate dextrose (ACD) by dissolving 85 mM Tri-sodium citrate, 71 mM citric acid, and 111 mM D-glucose in distilled water.</li>
	<li>Prepare a fixative solution by adding 2% (v/v) paraformaldehyde (PFA) to HEPES Tyrode&#39;s buffer, pH 7.4. Heat to dissolve (50 - 70 &#176;C) and set the pH to 7.4. Aliquot and store at -20 &#176;C. Thaw at 37 &#176;C before use.</li>
	<li>Prepare blocking buffer by dissolving 1% (m/v) human serum albumin (HSA) in HEPES Tyrode&#39;s buffer, pH 7.4</li>
	<li>Make a polyvinyl alcohol solution by adding 4.8 g of polyvinyl alcohol to 12 g of glycerol and mix well. Add 12 mL of distilled water and leave it on a rotator at room temperature overnight.
	<ol>
		<li>Add 24 mL of 0.2 M Tris-HCL, pH 8.5. Heat to 50 &#176;C while mixing for 30 min. Add 1.25 g of 1,4-diazabicyclo[2.2.2]octane (DABCO) and mix well. Centrifuge at 1,500 x g for 5 min. Aliquot the supernatant (1 mL is sufficient for 15-20 slides) and store at -20 &#176;C. Use the aliquots once.</li>
	</ol>
	</li>
</ol>
2. Cover Glass Preparation
<ol>
	<li>Degrease cover glasses (25 x 50 mm2) by incubating overnight in undiluted chromosulfuric acid. Rinse the cover glasses with distilled water and dry them overnight at 60 &#176;C in an oven.</li>
	<li>Adhere a strip of a paraffin film (10 x 15 cm2) on top of a laboratory bench with a drop of water and remove the air by smoothing the paraffin film. Pipette 80 &#181;L drops of 10 &#181;g/mL VWF or 100 &#181;g/mL fibrinogen onto the paraffin film. Place the glasses onto the drops; the protein solution will spread between the two surfaces to cover the whole back side of the glass. Incubate for 1.5 h at room temperature (RT).</li>
	<li>Remove the paraffin film and block the cover glasses by placing them, with the coated side up in a 4-well plate with blocking buffer. Incubate for 1 h at 37 &#176;C or overnight at 4 &#176;C. As a control, block a cover glass that has not been coated with VWF or fibrinogen.</li>
</ol>
3. Preparation of Platelet-rich Plasma (PRP)
<ol>
	<li>Collect citrated whole blood (final concentration: 0.32% sodium citrate (M/V)) by venipuncture.</li>
	<li>Centrifuge the blood for 15 min at 160 x g and RT without brake. Collect the supernatant (i.e., the PRP) with a plastic Pasteur pipette. 
	NOTE: After centrifugation, three layers can be observed (from top to bottom): PRP, white blood cells, and red blood cells. Make sure not to include the white and red cells. Typically, 3 mL of PRP can be collected after centrifuging of a 9-mL blood tube.</li>
	<li>Continue to section 4 if the experiment will be performed with washed platelets. If PRP is desired, continue with the next step.</li>
	<li>After PRP collection, centrifuge the remainder of the blood (containing the red cell pellet) again for 10 min at 2,000 x g without brake. Collect the supernatant containing the platelet-poor plasma (PPP). 
	NOTE: Typically, 2 mL can be collected after this second centrifugation step.</li>
	<li>Count the number of the platelets in the PRP on a cell analyzer and dilute it with the PPP (step 3.4) to a concentration of 150,000 platelets/&#181;L. 
	NOTE: The total volume of diluted PRP depends on the platelet count of the donor. Platelet counts vary widely between donors, and PRP dilution requires assessment on an individual basis. Platelet counts are considered normal when within 150,000 and 450,000 platelets/&#181;L.</li>
	<li>Continue to section 5.</li>
</ol>
4. Preparation of Washed Platelets
<ol>
	<li>Determine the mean platelet volume (MPV) in the PRP using a cell analyzer. 
	NOTE: This is an indicator of platelet pre-activation11. A normal MPV for resting platelets is 7.5-11.5 fL11. The MPV should not increase by more than 1.5 fL during the platelet washing procedure.</li>
	<li>Add a 1/10 volume of ACD to PRP to prevent platelet pre-activation and centrifuge for 15 min at 400 x g and RT without brake. Remove the supernatant and re-suspend the pellet carefully in HEPES Tyrode&#39;s buffer, pH 6.5, to the original volume of PRP.</li>
	<li>Thaw an aliquot of prostacyclin by adding TBS to a concentration of 10 &#181;g/mL; keep it on ice.</li>
	<li>To prevent platelet activation, add prostacyclin at a final concentration of 10 ng/mL to the re-suspended platelets and immediately centrifuge for 15 min at 400&#160;x g and RT without brake. Remove the supernatant and re-suspend the pellet carefully in HEPES Tyrode&#39;s buffer, pH 7.4.</li>
	<li>Count the number of platelets (step 3.5) and determine the change in MPV (step 4.1) (the MPV should not change more than 1.5 fL).</li>
	<li>Adjust the number of platelets with HEPES Tyrode&#39;s buffer, pH 7.4, to a concentration of 150,000 platelets/&#181;L. Leave the platelets to recover for 30 min at RT prior to the experiments. Use washed platelets for experiments within 4 h after isolation.</li>
</ol>
5. Flow Chamber Assembly
NOTE: These experiments make use of an in-house developed flow chamber12. A variety of commercially produced flow chambers can be used, as long as they can hold a cover glass, which preferentially is removable for further analyses. The flow chamber used for the described experiments is made from poly(methyl methacrylate) to precisely fit a microscope insert stage. It contains an inlet and outlet (Figure 1A-C; 1, 2), as well as a vacuum connection (Figure 1A and C; 3). A customized silicone sheet (Figure 1A and D; 4) is placed on top. Two cut-outs form vacuum channels (Figure 1A and D; 5) to attach the cover glass (Figure 1A; 6) tightly to the chamber. A central cut-out forms the flow channel (Figure 1A and D; 7; 2.0 mm width and 0.125 mm height). The inlet and outlet are connected to the flow channel, and the vacuum is connected to the vacuum channel.
<ol>
	<li>Cover the top of the flow chamber with distilled water and place the customized silicone sheet with the smooth side down on the water. Float the sheet into position by removing excess water while keeping the sheet in position. 
	NOTE: This silicone sheet is re-usable (silicone is known for its inert properties); generally, platelets have not been observed to attach to it. The sheets can be cleaned with detergent and re-used until the material becomes damaged (i.e., tearing, especially in the area between channels). Generally, a sheet can be used for 20 days with multiple experiments per day.</li>
	<li>Incubate the flow chamber for 1 h at 60 &#176;C to evaporate the residual water to firmly adhere the silicone sheet to the flow chamber.</li>
	<li>With a plastic Pasteur pipette, add a drop of HEPES Tyrode&#39;s buffer, pH 7.4, onto the flow channel. Place the cover glass (see section 2) with the coated side down, onto the flow channel. Attach the vacuum pump to the vacuum connection (Figures 1A and C; 3). Apply ~10 kPa pressure.</li>
</ol>
6. Pre-staining of the Platelets and Preparation of the Antibodies
<ol>
	<li>Platelet staining
	<ol>
		<li>Incubate the washed platelets with 10 &#181;g/mL DAPI for 30 min at 37 &#176;C in the dark. To wash away unbound DAPI and to prevent platelet activation, add prostacyclin at a final concentration of 10 ng/mL and immediately centrifuge for 15 min at 400 x g and RT without brake.</li>
		<li>Reconstitute the platelet pellet in HEPES Tyrode&#39;s buffer, pH 7.4, to a platelet concentration of 150,000 platelets/&#181;L.</li>
	</ol>
	</li>
	<li>Preparation of antibodies
	<ol>
		<li>Mix biotin-labeled antibody with Alexa-labeled streptavidin at a molar ratio of 1:1. Incubate for a minimum of 10 min at room temperature before use (step 7.6). 
		NOTE: Biotin labeling efficiency can vary between different batches. Should there be problems with the visualization of target molecules, control experiments should be performed to confirm successful biotinylation, as well the target-binding properties of the specific biotinylated antibody.</li>
	</ol>
	</li>
</ol>
7. Perfusion
<ol>
	<li>Start the fluorescence microscope, as well as the microscope software. Use the microscope settings listed in Table 1. 
	NOTE: Initiate the microscope and recording settings for the live-cell visualization and recording of the platelets and the chosen fluorescently labeled antibodies and/or dyes by loading an appropriate experiment in the microscope software. Perform flow experiments at RT.</li>
	<li>Block the inlet tubing by connecting a 10-mL syringe to the tubing and aspirating 1% HSA blocking buffer into the inlet tubing. Incubate for 15 min at RT. Rinse the inlet tubing by aspirating HEPES Tyrode&#39;s buffer, pH 7.4, into the inlet tubing.</li>
	<li>Connect the blocked inlet tubing and the untreated outlet tubing, both with a diameter of 1.02 mm and a ~20-30 cm length to the inlet and outlet of the chamber. Connect a 10-mL syringe (or lower volume for more accuracy) to the outlet tubing. Power on the syringe pump. 
	NOTE: The diameter of the syringe, in combination with the flow rate set on the pump, determine the volumetric flow rate. Together with the surface area of the flow channel, the shear rate can be calculated according to the formula13 below. Hence, it is possible to manipulate the shear rate by changing the flow rate of the syringe pump or altering the dimensions of the flow channel. A shear rate of 800-1,600 s-1 typically models for arterial blood flow, while 25 - 100s-1 models for venous blood flow. For this setup, a flow rate of 7.5 &#181;L/min results in a shear rate of 25 s-1. 
	Shear rate = 6Q / h2w in s-1 
	Wherein: Q = volumetric flow rate (mL/s), h = height channel (cm) (0.0125 cm; this is the thickness of the silicone sheet), w = width channel (cm) (0.2 cm in this chamber).</li>
	<li>Place a drop of immersion oil on a 100X objective (total amplification of 1,000X) and mount the chamber on the microscope with the glass surface down.</li>
	<li>Place the inlet tubing into a tube containing HEPES Tyrode&#39;s buffer, pH 7.4. Manually pull the plunger of the syringe connected to the outlet tubing and pull the solution through the chamber to remove the air from the system. Place the syringe into the syringe pump (Figure 1B and C; 8) and run the pump briefly to tighten the plunger and ensure stable flow.</li>
	<li>Add antibodies and/or dyes to the platelet suspension or PRP, carefully mix with a plastic pipette, and transfer the mixture to a 1.5-mL tube. 
	NOTE: For a typical 30-min perfusion at shear rate 25s-1 (7.5 &#181;L/min), a minimum of 225 &#181;L of platelet suspension is needed. The antibodies used for the representative results are shown in Table 2.</li>
	<li>Move the inlet tubing, while squeezing, from the HEPES Tyrode&#39;s buffer, pH 7.4, to a 1.5-mL tube containing either PRP or the washed platelets and the antibodies and/or dyes (Figure 1B and C; 9). 
	NOTE: It is important that, when transferring the tubing, the tubing is squeezed (pressing out the air) to prevent air from entering the tubing. It is possible to treat adherent platelets with a variety of reagents in a subsequent step by moving the inlet tubing to another tube in a similar manner. A list of potentially useful reagents is provided in Table 3.</li>
	<li>Select the &#34;live visualization&#34; option in the software, focus the microscope on the surface of the coated cover glass, and start the pump at a shear rate of 1,600s-1 (484 &#181;L/min) until the platelets reach the flow chamber. At this moment, reduce the shear rate back to 25s-1 by changing the settings of the syringe pump to a flow rate of 7.5 &#181;L/min.</li>
	<li>Monitor the surface of the coated side of the cover glass, wait until the first platelet starts to adhere, focus the microscope on this platelet, and start recording by initiating the experiment in the software. See Table 1 for the recording settings used here.</li>
	<li>Follow the live recording visually. After 30 min, stop the syringe pump. 
	NOTE: Ensure that the platelets stay in focus during the course of the experiment. Typically, 20-30 platelets attach in the field of view at a magnification of 1,000X; this is about 2,000-3,000 platelets in the whole flow channel.</li>
	<li>Fixation using the flow chamber (optional).
	<ol>
		<li>When the flow has stopped after the syringe pump is stopped, transfer the inlet tubing (air-free) to the fixative solution. Restart the pump and flow the fixation buffer through the channel for a minimum of 10 min at the same flow rate as mentioned in step 7.8.</li>
	</ol>
	</li>
	<li>Remove the chamber from the microscope, clean the immersion oil from the glass, disconnect the vacuum, and carefully remove the cover glass from the chamber with an 18 G needle. 
	NOTE: Prevent the cover glass from breaking and damaging the silicon sheet.</li>
	<li>Place the cover slide (with the cells directed upwards) in a 4-well plate on top of a tissue, pre-wetted with distilled water. 
	NOTE: This prevents the cover glass from adhering to the well surface and prevents drying.</li>
	<li>Pipette 750 &#181;L of fixative solution on top of the cover glass and incubate for 1 h at room temperature. In case the samples cannot be analyzed directly, store them in 0.5% PFA (diluted by HEPES Tyrode&#39;s buffer, pH 7.4) at 4 &#176;C to ensure stable fixation. Proceed to section 8 to process the cover glass for imaging.</li>
	<li>After use, rinse the chamber, tubing, and sheet with distilled water and incubate overnight in a detergent solution. Before the re-use of the chamber and other components, rinse with distilled water.</li>
	<li>Save the microscope raw data files containing all the metadata. When needed, export the movies to .MOV, h.264 compressed, or .AVI uncompressed. Save separate frames as JPEG or TIF files.</li>
</ol>
8. Sample Preparation for Confocal Fluorescence Microscopy
<ol>
	<li>Transfer the cover glasses to new 4-well plates and rinse 5 times with 3 mL of HEPES Tyrode&#39;s buffer, pH 7.4. Add 3 mL/well of blocking buffer and incubate for 1 h at RT.</li>
	<li>Remove the blocking buffer; add 3 mL/well of 0.15% glycine (M/V) in HEPES Tyrode&#39;s buffer, pH 7.4; and incubate for 15 min at RT.</li>
	<li>Remove glycine solution and wash 5 times in 3 mL/well of blocking buffer. Optionally, add fluorophore-conjugated antibodies diluted in blocking buffer for additional staining. Incubate for 1 h at RT in the dark.</li>
	<li>Wash 5 times with blocking buffer and 2 times with distilled water. Dry the glass by removing excess liquid with a tissue (from the edges inward), without touching the cells.</li>
	<li>Pipette 60 &#181;L of polyvinyl alcohol solution onto a microscope slide. Place the cover glass, with the cells down, on top of the drop and let the polyvinyl alcohol spread between the two surfaces. Incubate overnight at RT in the dark.</li>
	<li>Analyze the cells by confocal microscopy14.</li>
	<li>Save the raw data files of the recorded stack data containing all the metadata. When needed, process the raw data files into MOV, h.264 compressed, or .AVI uncompressed files. Save separate stacks as JPEG or TIF files.</li>
</ol>

<ol><li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((von+Willebrand+factor%2C+Jedi+knight+of+the+bloodstream%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">von Willebrand factor, Jedi knight of the bloodstream.</a> Blood. 124 (9), 1412-1425 (2014).</li>
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</ol>

The authors have nothing to disclose.

Worldwide, thrombosis is a leading cause of death and morbidity, and platelets play a central role in its development. This work describes a method for the live-cell imaging of platelet degranulation under flow. It is generally assumed that, when platelets become activated, all granular contents are directly released into solution. The accompanying results suggest that this is not necessarily the case. During adhesion and degranulation, platelets retain a significant amount of polyphosphate (Figure 4). A...

Platelets are anucleate cells that circulate in the blood stream. Their main function is to seal vascular breaches at sites of injury and to prevent blood loss. At these sites of injury, subendothelial collagen fibers become exposed and are subsequently covered by the multimeric protein, von Willebrand factor (VWF). VWF interacts with the platelets in circulation in a mechanism that depends upon the glycoprotein Ib&#945;-IX-V complex on the cell surface1, slowing down the speed of the platelets. This is particularly important at high shear rates. The platelets subsequently undergo morphological changes while receiving activating impulses from collagen. This leads to irreversible spreading and eventually to platelet aggregation. Both processes depend upon the secretion of granule contents to facilitate platelet-platelet crosstalk. Amongst others, platelet &#945;-granules contain fibrinogen and VWF to assist platelet adhesion and to bridge platelets together in an integrin-dependent manner. The platelet-dense granules contain inorganic compounds2, including calcium and adenosine diphosphate (ADP), which help to reinforce platelet activation. Furthermore, platelets contain mediators of (allergic) inflammation3, complement-controlling proteins4, and angiogenesis factors5,6, raising the questions of whether and how these contents are differentially released under varying conditions.
Since the 1980s, the study of platelet function in flow models has been valuable to the investigation of thrombotic mechanisms7. Since then, much technical progress has been made, and flow models that include fibrin formation are currently developed to assay the hemostatic potential of therapeutic platelet concentrates ex vivo8 or to investigate the influence of disturbances in shear rates on thrombus morphology9. The differences in the molecular and cell-biological mechanisms that drive stable adhesion and physiological thrombus formation (hemostasis) versus pathological thrombus formation (thrombosis) may be very subtle and motivate the development of flow models that allow for the real-time visualization of these subcellular processes.
An example of a process for which such a setup would be valuable is the (re)distribution of intracellular polyphosphate and the recruitment of clotting factors to uncover the time-dependent impact that this has on fibrin ultrastructure10. Studies are often limited to end-point analyses. The main aim of the described method is to enable the real-time visual investigation of dynamic subcellular processes that take place during platelet activation under flow.

<table><tbody><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)&amp;nbsp;</td><td>VWR</td><td>441476L</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>S-0876</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>31434</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>31248</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck KGaA</td><td>1.05886</td><td></td></tr><tr><td>D-glucose</td><td>Merck KGaA</td><td>1.04074</td><td></td></tr><tr><td>Prostacyclin&amp;nbsp;</td><td>Cayman Chemical</td><td>18220</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>Merck KGaA</td><td>1.06448</td><td></td></tr><tr><td>Citric acid&amp;nbsp;</td><td>Merck KGaA</td><td>1.00244</td><td></td></tr><tr><td>Cover glasses</td><td>Menzel-Gl&amp;auml;ser</td><td>BBAD02400500#A</td><td>24 x 50 mm, No. 1 = 0.13 - 0.16 mm thickness.</td></tr><tr><td>Chromosulfuric acid (2% CrO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Riedel de Haen</td><td>07404</td><td>CAS [65272-70-0].</td></tr><tr><td>Von Willebrand factor (VWF)</td><td>in-house purified</td><td></td><td></td></tr><tr><td>Fibrinogen</td><td>Enzyme Research Laboratories</td><td>FIB3L</td><td></td></tr><tr><td>4 well dish, non-treated</td><td>Thermo Scientific</td><td>267061</td><td></td></tr><tr><td>Human Serum Albumin Fraction V</td><td>Haem Technologies Inc.</td><td>823022</td><td></td></tr><tr><td>Blood collection tubes, 9 mL, 9NC Coagulation Sodium Citrate 3.2%</td><td>Greiner Bio-One</td><td>455322</td><td></td></tr><tr><td>Cell analyser&amp;nbsp;</td><td>Abbott Diagnostics</td><td>CELL-DYN hematology analyzer</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>30525-89-4&amp;nbsp;</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus, Holliston, MA</td><td>Harvard apparatus 22</td><td></td></tr><tr><td>10 mL syringe with 14.5 mm diameter</td><td>BD biosciences</td><td>305959</td><td>Luer-Lok syringe</td></tr><tr><td>Anti-CD63-biotin&amp;nbsp;</td><td>Abcam&amp;nbsp;</td><td>AB134331</td><td></td></tr><tr><td>Anti-CD62P-biotin&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>Dy137</td><td></td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td><td>Polysciences Inc.&amp;nbsp;</td><td>9224</td><td></td></tr><tr><td>Streptavidin, Alexa Fluor 488 conjugate</td><td>Thermo Scientific</td><td>S11223&amp;nbsp;</td><td></td></tr><tr><td>Immersion oil</td><td>Zeiss</td><td>444963-0000-000</td><td></td></tr><tr><td>Detergent solution</td><td>Unilever, Biotex</td><td></td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>56-40-6&amp;nbsp;</td><td></td></tr><tr><td>Polyvinyl alcohol</td><td>Sigma</td><td>9002-89-5</td><td>Mowiol 40-88.</td></tr><tr><td>Tris hydrochloride</td><td>Sigma</td><td>1185-53-1&amp;nbsp;</td><td></td></tr><tr><td>1,4-Diazabicyclo[2.2.2]octane (DABCO)</td><td>Sigma</td><td>280-57-9</td><td></td></tr><tr><td>Sheep Anti-hVWF pAb</td><td>Abcam&amp;nbsp;</td><td>AB9378</td><td></td></tr><tr><td>Alexa fluor 488-NHS</td><td>Thermo Scientific</td><td>A20000</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>15523-1L-R</td><td></td></tr><tr><td>Parafinn film</td><td>Bemis</td><td>PM-996</td><td>4 in. x 125 ft. Roll.</td></tr><tr><td>Silicone sheet non-reinforced</td><td>Nagor</td><td>NA 500-1</td><td>200 mm x 150 mm x 0.125 mm.</td></tr><tr><td>Customized cut silicone sheet with perfusion and vacuum channels</td><td>in-house made</td><td></td><td>Made of Silicone sheet non-reinforced (Nagor, NA 500-1)</td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf AG</td><td>T9661-1000AE</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss Observer Z1&amp;nbsp;</td><td></td><td>Equiped with LED excitation lights.</td></tr><tr><td>Microscope software</td><td>Zeiss ZEN 2</td><td>blue edition</td><td></td></tr><tr><td>18 G needle (18 G x 1 1/2&quot;)</td><td>BD biosciences</td><td>305196</td><td></td></tr><tr><td>NaCl</td><td>Riedel de Haen</td><td>31248375</td><td></td></tr><tr><td>Tris</td><td>Roche</td><td>10708976</td><td></td></tr><tr><td>Plastic pasteur pipet</td><td>VWR</td><td>612-1681</td><td>&amp;nbsp;7 mL non sterile, graduated up to 3 mL.</td></tr><tr><td>Silicone tubing</td><td>VWR</td><td>228-0656</td><td>Inner diameter. x Outer diameter x Wall thickness = 1.02 x 2.16 x 0.57 mm.</td></tr><tr><td>Microscope slides</td><td>Thermo Scientific</td><td>ABAA000001##12E</td><td>76 x 26 x 1 mm, ground edges 45 &amp;deg;, frosted end.</td></tr></tbody></table>

live-cell imaging of platelet degranulation and secretion under flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

Figure 1 shows images of the flow chamber and experimental setup; the position and dimensions of the silicon sheet; and tubing connections. Figure 2 provides details on the dimensions of the flow chamber. Figure 3 and Movie 1 show a time-series of images of platelet adhesion and spreading on immobilized VWF. CD63 is a transmembrane protein that is inserted into the membrane of intrac...

Watch this Scientific Journal Video about Live-cell Imaging of Platelet Degranulation and Secretion Under Flow at JoVE.com

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

live-cell-imaging-of-platelet-degranulation-and-secretion-under-flow

Research

JoVE Journal

Biology

11.6K Views.  University Medical Center Utrecht. This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Video: Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

Bioengineering

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Immunology and Infection

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known as leukocyte transendothelial migration. Two routes for leukocytes to cross the endothelial monolayer have been described: the paracellular route, i.e., through the cell-cell junctions and the transcellular route, i.e., through the endothelial cell body. However, it has been technically difficult to discriminate between the para- and transcellular route. We developed a simple in vitro assay to study the distribution of endogenous VE-cadherin and PECAM-1 during neutrophil transendothelial migration under physiological flow conditions. Prior to neutrophil perfusion, endothelial cells were briefly treated with fluorescently-labeled antibodies against VE-cadherin and PECAM-1. These antibodies did not interfere with the function of both proteins, as was determined by electrical cell-substrate impedance sensing and FRAP measurements. Using this assay, we were able to follow the distribution of endogenous VE-cadherin and PECAM-1 during transendothelial migration under flow conditions and discriminate between the para- and transcellular migration routes of the leukocytes across the endothelium.

Leukocytes cross the endothelial monolayer using the paracellular or the transcellular route. We developed a simple assay to follow the distribution of endogenous junctional VE-cadherin and PECAM-1 during leukocyte transendothelial migration under physiological flow to discriminate between the two transmigration routes.

During inflammation, leukocytes leave the circulation and cross the endothelium to fight invading pathogens in underlying tissues. This process is known ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted in the concept of the leukocyte adhesion cascade. However, the exact molecular mechanisms involved in leukocyte recruitment have not yet been fully identified. Since leukocyte recruitment remains an important subject in the field of infection, inflammation, and (auto-) immune research, we present a straightforward laminar flow-based assay to study underlying mechanisms of the adhesion, de-adhesion, and transmigration of leukocytes under venous and arterial flow regimes. The in vitro assay can be used to study the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners in different models of vascular inflammation. This protocol describes a laminar flow-based assay using a parallel-flow chamber and an inverted phase contrast microscope connected to a camera to study the interactions of leukocytes and endothelial cells or platelets, which can be visualized and recorded then analyzed offline. Endothelial cells, platelets, or leukocytes can be pretreated with inhibitors or antibodies to determine the role of specific molecules during this process. Shear conditions, i.e. arterial or venous shear stress, can be easily adapted by the viscosity and flow rate of the perfused fluids and the height of the channel.

Leukocytes avidly interact with vascular cells and platelets after vessel wall injury or during inflammation. Here, we describe a straightforward laminar flow-based assay to characterize the molecular mechanisms that underlie the interactions between leukocytes and their cellular partners.

The recruitment of leukocytes upon injury or inflammation to sites of injury or tissue damage has been investigated during recent decades and has resulted ...

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like pressure or mechanical perturbations. Mechanoreceptors detect their stimuli and transmit signals via a great diversity of mechanisms. Some of the most common roles for mechanoreceptors are in neuronal responses, like touch and pain, or hair cells which function in balance and hearing. Mechanosensation is also important for cell types which are regularly exposed to shear stress such as endothelial cells, which line blood vessels, or blood cells which experience shear in normal circulation. Viscometers are devices that detect the viscosity of fluids. Rotational viscometers may also be used to apply a known shear force to fluids. The ability of these instruments to introduce uniform shear to fluids has been exploited to study many biological fluids including blood and plasma. Viscometry may also be used to apply shear to the cells in a solution, and to test the effects of shear on specific ligand-receptor pairs. Here, we utilize cone-plate viscometry to test the effects of endogenous levels of shear stress on platelets treated with antibodies against the platelet mechanosensory receptor complex GPIb-IX.

We describe an in-solution method to apply uniform shear to platelet surface receptors using cone-plate viscometry. This method may also be used more broadly to apply shear to other cell types and cell-fragments and need not target a specific ligand-receptor pair.

Many biological cells/tissues sense the mechanical properties of their local environments via mechanoreceptors, proteins that can respond to forces like ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

Platform for Assessing Platelet Function in Trauma and Transfusion

Microfluidics in Assessing Platelet Function

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying aspects of thrombosis and hemostasis. At high-shear environments simulating arterial flow, a microfluidic assay facilitates the study of platelet function, as platelet-rich thrombi form in a localized stenotic region of a flow channel. Utilizing devices that allow for small sample volume can additionally aid in evaluating platelet function under flow from volume-limited patient samples or animal models. Studying trauma patient samples or samples following platelet product transfusion may aid in directing therapeutic strategies for patient populations in which platelet function is critical. Effects of platelet inhibition via pharmacological agents can also be studied in this model. The objective of this protocol is to establish a microfluidic platform that incorporates physiologic flow, biological surfaces, and relevant hemostatic mechanisms to assess platelet function with implications for the study of trauma induced coagulopathy and transfusion medicine.

Platelet function under flow can be assessed and simulated hemostatic resuscitation can be modeled using a microfluidic device, which has applications in trauma and transfusion medicine.

Microfluidics incorporate physiologically relevant substrates and flows that mimic the vasculature and are, therefore, a valuable tool for studying ...

Medicine

Analyzing Platelet Phosphatidylserine and Microvesicle Release

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and releasing PS-expressing microvesicles that retain the original membrane architecture and cytoplasmic components of their originating cells. The accessibility of phosphatidylserine facilitates the binding of major coagulation factors, significantly amplifying the catalytic efficiency of coagulation enzymes, while microvesicle release acts as a pivotal mediator of intercellular signaling. Procoagulant platelets play a crucial role in clot stabilization during hemostasis, and their increased proportion in the bloodstream correlates with an increased risk of thrombosis. It has also been shown that platelet microvesicles are rich in growth factors that promote wound healing and inflammatory modulation. Analyzing phosphatidylserine exposure and microvesicle release using flow cytometry poses significant challenges due to their small size and the limited number of positive events for markers of interest. Despite considerable advances in the last decade, methods for assessing phosphatidylserine exposure and microvesicle release remain a work in progress. Unfortunately, no single universally applicable protocol exists, and several factors must be evaluated to determine the most appropriate methodology for each specific application. Here, we describe a detailed protocol for isolating washed platelets from human blood, followed by collagen and/or thrombin activation, to measure the exposure of phosphatidylserine and microvesicle release that characterize procoagulant platelets. This protocol is designed to facilitate the initial preparation of platelet-rich plasma and the isolation of washed platelets. Finally, phosphatidylserine exposure and microvesicle release are quantified by flow cytometry, enabling the identification of procoagulant platelets.

Procoagulant platelet formation has been correlated with an increased risk of thrombosis. Presented here is a precise protocol for isolating washed platelets from human blood, intended to quantify the exposure of phosphatidylserine and microvesicle release, which are distinctive features of procoagulant platelets.

Activated platelets promote coagulation primarily by exposing the procoagulant phospholipid phosphatidylserine (PS) on their outer membrane surfaces and ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Summary

Explore More Videos

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Real-time Imaging of Endothelial Cell-cell Junctions During Neutrophil Transmigration Under Physiological Flow

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Laminar Flow-based Assays to Investigate Leukocyte Recruitment on Cultured Vascular Cells and Adherent Platelets

A Uniform Shear Assay for Human Platelet and Cell Surface Receptors via Cone-plate Viscometry

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Microfluidics in Assessing Platelet Function

Procoagulant Platelet Characterization by Measuring Phosphatidylserine Exposure and Microvesicle Release from Human Purified Platelets