Name: live-cell imaging of platelet degranulation and secretion under flow
Uploaded: 2017-07-10T10:00:00
Description: Watch this Scientific Journal Video about Live-cell Imaging of Platelet Degranulation and Secretion Under Flow at JoVE.com

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 vari...

<ol>
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L., Flaumenhaft, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+granule+exocytosis:+a+comparison+with+chromaffin+cells.">Platelet granule exocytosis: a comparison with chromaffin cells.</a> Front Endocrinol (Lausanne). 4, 11 (2013).</li><li>May, B., Menkens, I., Westermann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+release+of+serotonin+and+histamine+from+blood+platelets+of+the+rabbit+by+aliphatic+and+aromatic+amines.">Differential release of serotonin and histamine from blood platelets of the rabbit by aliphatic and aromatic amines.</a> Life Sci. 6 (19), 2079-2085 (1967).</li><li>Schmaier, A. H., Smith, P. M., Colman, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+C1-+inhibitor.+A+secreted+alpha-granule+protein.">Platelet C1- inhibitor. A secreted alpha-granule protein.</a> J. Clin. Invest. 75 (1), 242-250 (1985).</li><li>Battinelli, E. M., Markens, B. A., Italiano, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+angiogenesis+regulatory+proteins+from+platelet+alpha+granules:+modulation+of+physiologic+and+pathologic+angiogenesis.">Release of angiogenesis regulatory proteins from platelet alpha granules: modulation of physiologic and pathologic angiogenesis.</a> Blood. 118 (5), 1359-1369 (2011).</li><li>Kamykowski, J., Carlton, P., Sehgal, S., Storrie, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+immunofluorescence+mapping+reveals+little+functional+coclustering+of+proteins+within+platelet+&#945;-granules.">Quantitative immunofluorescence mapping reveals little functional coclustering of proteins within platelet &#945;-granules.</a> Blood. 118 (5), 1370-1373 (2011).</li><li>Sakariassen, K. S., Aarts, P. A., de Groot, P. G., Houdijk, W. P., Sixma, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perfusion+chamber+developed+to+investigate+platelet+interaction+in+flowing+blood+with+human+vessel+wall+cells,+their+extracellular+matrix,+and+purified+components.">A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components.</a> J Lab Clin Med. 102 (4), 522-535 (1983).</li><li>Van Aelst, B., Feys, H. B., Devloo, R., Vandekerckhove, P., Compernolle, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+Flow+Chambers+Using+Reconstituted+Blood+to+Model+Hemostasis+and+Platelet+Transfusion+In+Vitro.">Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro.</a> J Vis Exp. (109), (2016).</li><li>Nesbitt, W. S., Westein, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+shear+gradient-dependent+platelet+aggregation+mechanism+drives+thrombus+formation.">A shear gradient-dependent platelet aggregation mechanism drives thrombus formation.</a> Nat Med. 15 (6), 665-673 (2009).</li><li>Mitchell, J. L., Lionikiene, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyphosphate+colocalizes+with+factor+XII+on+platelet-bound+fibrin+and+augments+its+plasminogen+activator+activity.">Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity.</a> Blood. 128 (24), 2834-2845 (2016).</li><li>Park, Y., Schoene, N., Harris, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mean+platelet+volume+as+an+indicator+of+platelet+activation:+methodological+issues.">Mean platelet volume as an indicator of platelet activation: methodological issues.</a> Platelets. 13 (5-6), 301-306 (2002).</li><li>Sixma, J. J., de Groot, P. G., van Zanten, H., IJsseldijk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+perfusion+chamber+to+detect+platelet+adhesion+using+a+small+volume+of+blood.">A new perfusion chamber to detect platelet adhesion using a small volume of blood.</a> Thromb Res. 92, S43-S46 (1998).</li><li>Slack, S. M., Turitto, V. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+chambers+and+their+standardization+for+use+in+studies+of+thrombosis.+On+behalf+of+the+Subcommittee+on+Rheology+of+the+Scientific+and+Standardization+Committee+of+the+ISTH.">Flow chambers and their standardization for use in studies of thrombosis. On behalf of the Subcommittee on Rheology of the Scientific and Standardization Committee of the ISTH.</a> Thromb Haemost. 72 (5), 777-781 (1994).</li><li>Oreopoulos, J., Berman, R., Browne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spinning-disk+confocal+microscopy:+present+technology+and+future+trends.">Spinning-disk confocal microscopy: present technology and future trends.</a> Methods Cell Biol. 123, 153-175 (2014).</li><li>Nishibori, M., Cham, B., McNicol, A., Shalev, A., Jain, N., Gerrard, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+CD63+is+in+platelet+dense+granules,+is+deficient+in+a+patient+with+Hermansky-Pudlak+syndrome,+and+appears+identical+to+granulophysin.">The protein CD63 is in platelet dense granules, is deficient in a patient with Hermansky-Pudlak syndrome, and appears identical to granulophysin.</a> J. Clin. Invest. 91 (4), 1775-1782 (1993).</li><li>Ruiz, F. A., Lea, C. R., Oldfield, E., Docampo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+platelet+dense+granules+contain+polyphosphate+and+are+similar+to+acidocalcisomes+of+bacteria+and+unicellular+eukaryotes.">Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes.</a> J. Biol. Chem. 279 (43), 44250-44257 (2004).</li><li>Tersteeg, C., Fijnheer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+von+Willebrand+Factor+under+Control:+Alternatives+for+ADAMTS13.">Keeping von Willebrand Factor under Control: Alternatives for ADAMTS13.</a> Semin Thromb Hemost. 42 (1), 9-17 (2016).</li><li>Tersteeg, C., de Maat, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmin+cleavage+of+von+Willebrand+factor+as+an+emergency+bypass+for+ADAMTS13+deficiency+in+thrombotic+microangiopathy.">Plasmin cleavage of von Willebrand factor as an emergency bypass for ADAMTS13 deficiency in thrombotic microangiopathy.</a> Circulation. 129 (12), 1320-1331 (2014).</li><li>Basir, A., de Groot, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Hemocompatibility+Testing+of+Dyneema+Purity+Fibers+in+Blood+Contact.">In Vitro Hemocompatibility Testing of Dyneema Purity Fibers in Blood Contact.</a> Innovations (Phila). 10 (3), 195-201 (2015).</li></ol>

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 ...

The overall goal of this experiment is to track platelet degranulation and secretion under flow using live-cell imaging. This method can help to answer key questions in the field of platelet biology. The main advantage of this technique is that it enables a versatile investigation of platelet contents at the subcellular level in live cells.Though this method can provide insight into platelet function, it can also be applied to other cell types such as mast cells. In general, individuals new to this method, will struggle because of problems with platelet isolation in a form of preactivation. We first had the idea for this method when we realized that platelet secretion could only be evaluated at end point stages.Real time images provide information of the release kinetics of platelet granular release. The procedure of platelet isolation will be demonstrated by Silvia Pignatelli, a student in our institute. Next, Arjan Barendrecht will demonstrate the real-time imaging experiments.To begin, degrease cover glasses by incubating them overnight in undiluted chromosulfuric acid. The next day, rinse them with distilled water. Then place then overnight at 60 degrees Celsius in an oven, to dry.Next, place a drop of water on a laboratory bench and adhere a 10 centimeter by 15 centimeter strip of a paraffin film on top of the drop by removing the air between the bench and the film. Then pipette 80 microliter drops of 10 microgram per milliliter von Willebrand factor or 100 microgram per milliliter fibrinogen onto the paraffin film. Place the dry cover glasses onto the drops and incubate them for one and a half hours at room temperature.Next, add three milliliters of blocking buffer into each well of a four well plate. Transfer the cover glasses with the coated side up into individual wells of the plate. Incubate them overnight at four degrees Celsius.Centrifuge nine milliliters of citrated whole blood for 15 minutes at 160 time G without a break. Then use a plastic Pasteur pipette to collect the supernatant containing the platelet-rich plasma. Add a one part of acid citrate dextrose to nine parts of the platelets by volume to prevent platelet preactivation.Then centrifuge the platelets for 15 minutes at 400 times G without a break. Next, remove the supernatant, leaving a platelet pellet. Then resuspend the pellet carefully in HEPES Tyrode buffer at a pH of 6.5 back to the original volume of the platelet sample.Then, add Prostacyclin to the resuspended platelets. Immediately centrifuge the sample and then remove the supernatant. Resuspend the pellet carefully in HEPES Tyrode buffer at a pH of 7.4.Next, place the washed platelets into a cell analyzer and count the number of platelets. Adjust the number of platelets in the sample to a concentration of 150, 000 platelets per microliter with HEPES Tyrode buffer at a pH of 7.4. After resuspension, leave the platelets to recover for 30 minutes at room temp prior to the experiment.Cover the top of the flow chamber with distilled water and place a 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. Incubate the flow chamber for one hour at 60 degrees Celsius to evaporate the residual water and to firmly adhere the silicone sheet to the flow chamber.With the plastic Pasteur pipette, add a drop of HEPES Tyrode buffer at a pH of 7.4 onto the flow channel. Then place the prepared cover glass with the coated side down, onto the flow channel. Turn on the vacuum pump to a pressure of around 10 kilopascals and attach the vacuum pump to the vacuum connection of the flow chamber.Add 10 micrograms per milliliter DAPI stain to the washed platelets, and wrap the tube containing the platelets in aluminum foil. Then incubate them for 30 minutes at 37 degrees Celsius. To prevent platelet activation, add prostacyclin at a final concentration of 10 nanograms per milliliter and immediately centrifuge the platelets for 15 minutes at 400 times G without using the brake to remove unbound DAPI.Reconstitute the platelet pellet and HEPES Tyrode buffer at a pH of 7.4 to the original volume with a concentration of 150, 000 platelets per microliter. Then, turn on the fluorescence microscope and load the microscope software. Set up the imaging parameters as described in table one of the accompanying text protocol.Block the inlet tubing by connecting a 10 milliliter syringe and aspirating blocking buffer into the inlet tubing. Block for 15 minutes at room temperature and then rinse the inlet tubing by aspirating HEPES Tyrode buffer 7.4 into the inlet tubing. Connect the blocked inlet tubing and the untreated outlet tubing to the inlet and outlet of the chamber.Then connect the 10 milliliter syringe to the outlet tubing. Place a drop of immersion oil on a 100 times objective and mount the chamber on the microscope with the glass surface facing downwards. Next, place the inlet tubing into a tube containing HEPES Tyrode buffer at a pH of 7.4.Manually pull the plunger of the syringe connected to the outlet tubing to pull the solution through the chamber and remove the air from the system. Then place the syringe into the syringe pump and run the pump briefly to tighten the plunger and ensure stable flow. With the flow system now ready, add the antibodies and or dyes to the platelet suspension and mix carefully.Then squeeze the inlet tubing and move it from the buffer to the tube containing the labeled platelets. Select live visualization in the software. Focus the microscope on the cover glass surface and start the pump at 484 microliters per minute.Run the pump until the platelets reach the flow chamber. Next, reduce the flow rate to 7.5 microliters per minute. Monitor the cover glass surface.Once the first platelet starts to adhere, focus the microscope on this platelet and start recording by initiating the experiment in the software. Record for 30 minutes and then stop the syringe pump. Save the microscope raw data files containing all the metadata.When needed, export the movies to the MOV H 264 compressed or AVI uncompressed. Save separate frames as JPEG or TIF files. When finished imaging, 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 gauge needle.Place the cover slide with the cells directed upwards in a new four well plate on top of a tissue prewetted with distilled water. Then pipette 750 microliters of a fixative solution on top of the cover glass and incubate it for one hour at room temperature in the dark. In case the samples cannot be analyzed directly, store them in 0.5%paraformaldehyde at four degrees Celsius to ensure stable fixation.This shows the time series of platelet adhesion and spreading on glass slides coated with immobilized von Willebrand factor. Shown in green in CD63, a transmembrane protein which is mobilized onto the platelet surface in a time-dependent manner. Similarly, shown in orange, P-selectin is also a transmembrane protein inserted in the membrane of intracellular alpha-granules arresting platelets.Following a flow experiment, confocal fluorescence microscopy can be used to produce a tilt series of the platelet adhesion. Seen here, CD63 is mainly located at the central granulomere while P-selectin is distributed over the entire cell body and appears concentrated at the edges. In these experiments, platelets, which do not have nuclear DNA, were preincubated with DAPI, which appears blue.In this case, DAPI stains the polyphosphates and dense granules. Appearing later as green staining, is the time dependent mobilization of CD63 onto the platelet surface. Despite clear signs of dense granule release, single cell analysis shows that polyphosphate is retained within or on the outside of these platelets.Once mastered, this technique can be done in six hours for four individual flow experiments. While attempting this procedure, it's important to prevent platelet preactivation. This can be achieved by careful manipulation of the cell suspension.Following this procedure, other methods like confocal microscopy can be used to obtain additional insight into the three dimensional distribution of target molecules. After watching this video, you should have a good understanding of how to study platelet function under flow at a single cell level.

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