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>

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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 border="0" cellpadding="0" cellspacing="0" width="1340">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)&#160;</td>
			<td style="width:295px;">VWR</td>
			<td style="width:227px;">441476L</td>
			<td style="width:372px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td style="width:295px;">Sigma</td>
			<td style="width:227px;">S-0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td style="width:295px;">Sigma</td>
			<td style="width:227px;">31434</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td style="width:295px;">Sigma</td>
			<td style="width:227px;">31248</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">MgSO4</td>
			<td style="width:295px;">Merck KGaA</td>
			<td style="width:227px;">1.05886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td style="width:295px;">Merck KGaA</td>
			<td style="width:227px;">1.04074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prostacyclin&#160;</td>
			<td style="width:295px;">Cayman Chemical</td>
			<td style="width:227px;">18220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tri-sodium citrate</td>
			<td style="width:295px;">Merck KGaA</td>
			<td style="width:227px;">1.06448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric acid&#160;</td>
			<td style="width:295px;">Merck KGaA</td>
			<td style="width:227px;">1.00244</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glasses</td>
			<td style="width:295px;">Menzel-Gl&#228;ser</td>
			<td style="width:227px;">BBAD02400500#A</td>
			<td>24x50mm, No. 1 = 0.13-0.16 mm thickness.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:447px;">Chromosulfuric acid (2% CrO3)</td>
			<td style="width:295px;">Riedel de Haen</td>
			<td>07404</td>
			<td>CAS [65272-70-0].</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Von Willebrand factor (VWF)</td>
			<td style="width:295px;">in-house purified</td>
			<td style="width:227px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fibrinogen</td>
			<td style="width:295px;">Enzyme Research Laboratories</td>
			<td style="width:227px;">FIB3L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 well dish, non-treated</td>
			<td style="width:295px;">Thermo Scientific</td>
			<td style="width:227px;">267061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Serum Albumin Fraction V</td>
			<td style="width:295px;">Haem Technologies Inc.</td>
			<td style="width:227px;">823022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blood collection tubes, 9 ml, 9NC Coagulation Sodium Citrate 3.2%</td>
			<td style="width:295px;">Greiner Bio-One</td>
			<td style="width:227px;">455322</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell analyser&#160;</td>
			<td style="width:295px;">Abbott Diagnostics</td>
			<td style="width:227px;">CELL-DYN hematology analyzer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td style="width:295px;">Sigma</td>
			<td>30525-89-4&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Syringe pump</td>
			<td style="width:295px;">Harvard Apparatus, Holliston, MA</td>
			<td style="width:227px;">Harvard apparatus 22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL syringe with 14.5 mm diameter</td>
			<td style="width:295px;">BD biosciences</td>
			<td style="width:227px;">305959</td>
			<td>Luer-Lok syringe</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Anti-CD63-biotin&#160;</td>
			<td style="width:295px;">Abcam&#160;</td>
			<td style="width:227px;">AB134331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Anti-CD62P-biotin&#160;</td>
			<td style="width:295px;">R&#38;D Systems</td>
			<td style="width:227px;">Dy137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#8217;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td style="width:295px;">Polysciences Inc.&#160;</td>
			<td style="width:227px;">9224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin, Alexa Fluor 488 conjugate</td>
			<td>Thermo Scientific</td>
			<td>S11223&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immersion oil</td>
			<td>Zeiss</td>
			<td>444963-0000-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Detergent solution</td>
			<td>Unilever, Biotex</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Sigma</td>
			<td>56-40-6&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyvinyl alcohol</td>
			<td style="width:295px;">Sigma</td>
			<td>9002-89-5</td>
			<td>Mowiol 40-88.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris hydrochloride</td>
			<td style="width:295px;">Sigma</td>
			<td>1185-53-1&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1,4-Diazabicyclo[2.2.2]octane (DABCO)</td>
			<td style="width:295px;">Sigma</td>
			<td>280-57-9</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sheep Anti-hVWF pAb</td>
			<td>Abcam&#160;</td>
			<td style="width:227px;">AB9378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Alexa fluor 488-NHS</td>
			<td style="width:295px;">Thermo Scientific</td>
			<td style="width:227px;">A20000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:227px;">15523-1L-R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Parafinn film</td>
			<td style="width:295px;">Bemis</td>
			<td style="width:227px;">PM-996</td>
			<td>4 in. x 125 ft. Roll.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Silicone sheet non-reinforced</td>
			<td style="width:295px;">Nagor</td>
			<td style="width:227px;">NA 500-1</td>
			<td>200mmx150mmx0.125mm.</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:447px;">Customized cut silicone sheet with perfusion and vacuum channels</td>
			<td style="width:295px;">in-house made</td>
			<td style="width:227px;"></td>
			<td>Made of Silicone sheet non-reinforced (Nagor, NA 500-1)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">1.5 mL tubes</td>
			<td style="width:295px;">Eppendorf AG</td>
			<td style="width:227px;">T9661-1000AE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Fluorescent microscope</td>
			<td style="width:295px;">Zeiss Observer Z1&#160;</td>
			<td style="width:227px;"></td>
			<td>Equiped with LED excitation lights.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Microscope software</td>
			<td style="width:295px;">Zeiss ZEN 2</td>
			<td style="width:227px;">blue edition</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">18 G needle (18 G x 1 1/2&#34;)</td>
			<td style="width:295px;">BD biosciences</td>
			<td>305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">NaCl</td>
			<td style="width:295px;">Riedel de Haen</td>
			<td style="width:227px;">31248375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Tris</td>
			<td style="width:295px;">Roche</td>
			<td style="width:227px;">10708976</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Plastic pasteur pipet</td>
			<td style="width:295px;">VWR</td>
			<td style="width:227px;">612-1681</td>
			<td>&#160;7 ml non sterile, graduated up to 3ml.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Silicone tubing</td>
			<td style="width:295px;">VWR</td>
			<td style="width:227px;">228-0656</td>
			<td>Inner diamete. x Outer diameter x Wall thickness = 1.02 x 2.16 x 0.57 mm.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Microscope slides</td>
			<td style="width:295px;">Thermo Scientific</td>
			<td style="width:227px;">ABAA000001##12E</td>
			<td>76 x 26 x 1 mm, ground edges 45&#176;, 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

11.5K 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

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

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 ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

LDL Cholesterol Uptake Assay Using Live Cell Imaging Analysis with Cell Health Monitoring

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic disorder, cardiovascular disease, and kidney disease. Currently, there is no available method to assess LDL uptake while simultaneously monitoring for health of the cells. The current study presents a protocol, using a live cell imaging analysis system, to acquire serial measurements of LDL influx with concurrent monitoring for cell health. This novel technique is tested in three human cell lines (hepatic, renal tubular epithelial, and coronary artery endothelial cells) over a four-hour time course. Moreover, the sensitivity of this technique is validated with well-known LDL uptake inhibitors, Dynasore and recombinant PCSK9 protein, as well as by an LDL uptake promoter, Simvastatin. Taken together, this method provides a medium-to-high throughput platform for simultaneously screening pharmacological activity as well as monitoring of cell morphology, hence cytotoxicity of compounds regulating LDL influx. The analysis can be used with different imaging systems and analytical software.

This protocol provides an efficient approach to measuring LDL cholesterol uptake with real time influx rates using a live cell imaging system in various cell types. This technique provides a platform to screen the pharmacological activity of compounds affecting LDL influx while monitoring for cell morphology and hence potential cytotoxicity.

The regulation of LDL cholesterol uptake through LDLR-mediated endocytosis is an important area of study in various major pathologies including metabolic ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...