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 anerkender finansiel støtte fra International Patient Organization for C1-Inhibitor Deficiencies (HAEi), Stichting Vrienden van Het UMC Utrecht og Landsteiner Foundation for Blood Transfusion Research (LSBR).

Det Lokale Medicinske Etiske Udvalg ved Universitetsmedicinsk Center Utrecht godkendte tegningen af ​​blod til ex vivo forskningsformål, herunder undersøgelserne. 1. Løsning Præparat <ol><li> Forbered en 4- (2-hydroxyethyl) -1-piperazinethansulfonsyre (HEPES) Tyrodes buffer ved at opløse 10 mM HEPES, 0,5 mM Na2 HPO4, 145 mM NaCl, 5 mM KCI, 1 mM MgSO4 og 5,55 mM D- Glucose i destilleret vand. <ol><li> Lav to varianter af bufferen: Indstil pH-niveauerne ved...

<ol>
	<li>Springer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=von+Willebrand+factor,+Jedi+knight+of+the+bloodstream.">von Willebrand factor, Jedi knight of the bloodstream.</a> Blood. 124 (9), 1412-1425 (2014).</li><li>Fitch-Tewfik, J. 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. 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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>

Globalt er trombose en ledende dødsårsag og morbiditet, og blodplader spiller en central rolle i udviklingen. Dette værk beskriver en metode til live-celle billeddannelse af blodplade degranulation under flow. Det antages generelt, at når blodplader bliver aktiveret, frigives alle granulære indhold direkte til opløsning. De medfølgende resultater tyder på, at dette ikke nødvendigvis er tilfældet. Under adhæsion og degranulering bevarer blodplader en signifikant mængde polyphosphat ( figur 4 ...

Blodplader er anukleatceller, som cirkulerer i blodstrømmen. Deres vigtigste funktion er at forsegle vaskulære brud på steder for skade og for at forhindre blodtab. På disse steder af skade bliver subendotelcollagenfibre eksponeret og bliver derefter dækket af det multimere protein, von Willebrand-faktor (VWF). VWF interagerer med blodpladerne i omløb i en mekanisme, der afhænger af glycoprotein Ibα-IX-V-komplekset på celleoverfladen 1 , hvilket nedsætter blodpladernes hastighed. Dette er især vigtigt ved høje forskydninger. Blodpladerne undergår efterfølgende morfologiske ændringer under modtagelse af aktiverende impulser fra collagen. Dette fører til irreversibel spredning og til sidst til blodpladeaggregering. Begge processer afhænger af udskillelsen af ​​granulindholdet for at lette trombocyt-blodplade-blodkroget. Bl.a. indeholder blodplade-a-granuler fibrinogen og VWF for at hjælpe blodpladeadhæsion og broBlodplader sammen på en integrinafhængig måde. De blodpladetætte granuler indeholder uorganiske forbindelser 2 , herunder calcium og adenosindiphosphat (ADP), som hjælper til at forstærke blodpladeaktivering. Desuden indeholder blodplader mediatorer af (allergisk) inflammation 3 , komplementstyrende proteiner 4 og angiogenesefaktorer 5 , 6 , der rejser spørgsmålene om, hvorvidt og hvordan disse indhold forskelligt frigives under forskellige betingelser. Siden 1980&#39;erne har undersøgelsen af ​​blodpladefunktion i flowmodeller været værdifuld for undersøgelsen af ​​trombotiske mekanismer 7 . Siden da er der sket meget teknisk fremgang, og flowmodeller, der indbefatter fibrindannelse, udvikles for øjeblikket til at analysere det hæmostatiske potentiale af terapeutiske blodpladekoncentrater ex vivo 8 eller tilUndersøg indflydelsen af ​​forstyrrelser i forskydningshastigheder på trombusmorfologi 9 . Forskellene i de molekylære og cellebiologiske mekanismer, der kører stabil vedhæftning og fysiologisk tromb dannelse (hæmostase) i forhold til patologisk trombusdannelse (trombose) kan være meget subtile og motivere udviklingen af ​​flowmodeller, der muliggør real-time visualisering af disse subcellulære processer. Et eksempel på en proces, for hvilken en sådan opsætning ville være værdifuld, er (re) fordeling af intracellulært polyphosphat og rekruttering af koagulationsfaktorer for at afdække den tidsafhængige virkning, som dette har på fibrin-ultrastruktur 10 . Undersøgelser er ofte begrænset til slutpunktsanalyser. Hovedformålet med den beskrevne metode er at muliggøre den realtidsvisuelle undersøgelse af dynamiske subcellulære processer, der finder sted under blodpladeaktivering under strømning.

<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

Blodplader er vigtige spillere i hæmostase, dannelsen af ​​trombier for at forsegle vaskulære brud. De er også involveret i trombose, dannelsen af ​​trombier, der fører til vaskulaturen og skadelige organer med livstruende konsekvenser. Dette motiverer videnskabelig forskning om trombocytfunktion og udvikling af metoder til at spore cellebiologiske processer, som de forekommer under strømningsbetingelser. En række strømningsmodeller er tilgængelige til undersøgelse af trombocytadhæsion og aggregering, to nøglefænomener i blodpladebiologi. Dette værk beskriver en metode til at studere realtids blodplade-degranulation under flow under aktivering. Metoden gør brug af et flowkammer koblet til en sprøjtepumpeopsætning, der er placeret under et bredt felt, inverteret LED-baseret fluorescensmikroskop. Opstillingen beskrevet her muliggør samtidig excitation af flere fluoroforer, der leveres af fluorescensmærkede antistoffer eller fluorescensEnt farvestoffer. Efter levende celledannelsesforsøg kan dækglasene behandles yderligere og analyseres ved hjælp af statisk mikroskopi ( dvs. konfokal mikroskopi eller scanningselektronmikroskopi).

Figur 1 viser billeder af strømningskammeret og eksperimentelle opsætninger; Positionen og dimensionerne af siliciumpladen; Og rørforbindelser. Figur 2 giver detaljer om dimensioner af strømningskammeret. Figur 3 og film 1 viser en tidsserie af billeder af trombocytadhæsion og spredning på immobiliseret VWF. CD63 er et transmembranprotein, der indsættes i membranen af ​​i...

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

Dette værk beskriver en fluorescensmikroskopi-baseret metode til undersøgelse af trombocytadhæsion, spredning og sekretion under strømning. Denne alsidige platform muliggør undersøgelse af trombocytfunktion til mekanisk undersøgelse af trombose og hæmostase.

Levende cellebilleddannelse af blodpladegravulation og sekretion under strømning

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

Research

JoVE Journal

Biology

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 ...

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

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 ...

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

To-og tre-dimensionelle Live-Cell Imaging af DNA skade responset Proteiner

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

Levende Cell Imaging af Early Autophagy begivenheder: 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 ...

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

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

Undersøgelse af Protein Kvalitetskontrol System af<em&gt; D. discoideum</em&gt; Brug Temperatur-kontrollerede 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 ...

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

Blandet Primære kulturer af murine Tyndtarm Beregnet for Studiet af Gut hormon sekretion og levende celler af enteroendokrine Cells

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

LDL-kolesterol optagelse Assay ved hjælp af levende celle Imaging analyse med celle sundhedsovervågning

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

Anvendelse af membran og cellevæg selektive fluorescerende farvestoffer til levende celle billeder af filamentøse svampe

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

Super-Resolution Live Cell Imaging of Subcellular Structures

Dynamisk cellebilledbehandling i superopløsning af undercellulære strukturer

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...