Name: proplatelet formation dynamics of mouse fresh bone marrow explants
Uploaded: 2021-05-20T15:00:00
Description: Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

De auteurs willen Jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert bedanken voor de technische bijstand. Dit werk werd ondersteund door ANR (Agence National de la Recherche) Grant ANR-17-CE14-0001-01 en ANR-18-CE14-0037.

Alle dierproeven werden uitgevoerd in overeenstemming met de Europese normen 2010/63/EU en het CREMEAS Committee on the Ethics of Animal Experiments van de Universiteit van Straatsburg (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg).
1. Bereiding van reagentia
<ol><li>Reagentia bereiden zoals beschreven in tabel 1.<ol>
<li>Los voor de Stock I elk poeder afzonderlijk op. Zorg ervoor dat de osmolariteit van het preparaat hoger is dan 295 mOsm/L....

<ol>
	<li>Thiery, J. P., Bessis, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+platelet+genesis;+in+vitro+study+by+cinemicrophotography.">Mechanism of platelet genesis; in vitro study by cinemicrophotography.</a> Reviews in Hematology. 11 (2), 162-174 (1956).</li><li>Becker, R. P., De Bruyn, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transmural+passage+of+blood+cells+into+myeloid+sinusoids+and+the+entry+of+platelets+into+the+sinusoidal+circulation:+A+scanning+electron+microscopic+investigation.">The transmural passage of blood cells into myeloid sinusoids and the entry of platelets into the sinusoidal circulation: A scanning electron microscopic investigation.</a> American Journal of Anatomy. 145 (2), 183-205 (1976).</li><li>Muto, 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+scanning+and+transmission+electron+microscopic+study+on+rat+bone+marrow+sinuses+and+transmural+migration+of+blood+cells.">A scanning and transmission electron microscopic study on rat bone marrow sinuses and transmural migration of blood cells.</a> Archivum Histologicum Japonicum. 39 (1), 51-66 (1976).</li><li>Cramer, E. M., 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=Ultrastructure+of+platelet+formation+by+human+megakaryocytes+cultured+with+the+Mpl+ligand.">Ultrastructure of platelet formation by human megakaryocytes cultured with the Mpl ligand.</a> Blood. 89 (7), 2336-2346 (1997).</li><li>Kaushansky, K., 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=Promotion+of+megakaryocyte+progenitor+expansion+and+differentiation+by+the+c-Mpl+ligand+thrombopoietin.">Promotion of megakaryocyte progenitor expansion and differentiation by the c-Mpl ligand thrombopoietin.</a> Nature. 369 (6481), 568-571 (1994).</li><li>Strassel, C., 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=Hirudin+and+heparin+enable+efficient+megakaryocyte+differentiation+of+mouse+bone+marrow+progenitors.">Hirudin and heparin enable efficient megakaryocyte differentiation of mouse bone marrow progenitors.</a> Experimental Cell Research. 318 (1), 25-32 (2012).</li><li>Aguilar, A., 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=Importance+of+environmental+stiffness+for+megakaryocyte+differentiation+and+proplatelet+formation.">Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.</a> Blood. 128 (16), 2022-2032 (2016).</li><li>Scandola, C., 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=Use+of+electron+microscopy+to+study+megakaryocytes.">Use of electron microscopy to study megakaryocytes.</a> Platelets. 31 (5), 589-598 (2020).</li><li>Eckly, A., 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=Characterization+of+megakaryocyte+development+in+the+native+bone+marrow+environment.">Characterization of megakaryocyte development in the native bone marrow environment.</a> Methods in Molecular Biology. 788, 175-192 (2012).</li><li>Eckly, A., 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=Abnormal+megakaryocyte+morphology+and+proplatelet+formation+in+mice+with+megakaryocyte-restricted+MYH9+inactivation.">Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation.</a> Blood. 113 (14), 3182-3189 (2009).</li><li>Eckly, A., 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=Proplatelet+formation+deficit+and+megakaryocyte+death+contribute+to+thrombocytopenia+in+Myh9+knockout+mice.">Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice.</a> Journal of Thrombosis and Haemostasis. 8 (10), 2243-2251 (2010).</li><li>Ortiz-Rivero, 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=C3G,+through+its+GEF+activity,+induces+megakaryocytic+differentiation+and+proplatelet+formation.">C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation.</a> Cell Communication and Signaling. 16 (1), 101 (2018).</li><li>Junt, T., 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=Dynamic+visualization+of+thrombopoiesis+within+bone+marrow.">Dynamic visualization of thrombopoiesis within bone marrow.</a> Science. 317 (5845), 1767-1770 (2007).</li><li>Zhang, Y., 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=Mouse+models+of+MYH9-related+disease:+mutations+in+nonmuscle+myosin+II-A.">Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.</a> Blood. 119 (1), 238-250 (2012).</li><li>Malara, A., 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=Extracellular+matrix+structure+and+nano-mechanics+determine+megakaryocyte+function.">Extracellular matrix structure and nano-mechanics determine megakaryocyte function.</a> Blood. 118 (16), 4449-4453 (2011).</li><li>Balduini, A., 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=Adhesive+receptors,+extracellular+proteins+and+myosin+IIA+orchestrate+proplatelet+formation+by+human+megakaryocytes.">Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.</a> Journal of Thrombosis and Haemostasis. 6 (11), 1900-1907 (2008).</li></ol>

Hier beschrijven we een eenvoudige en goedkope in vitro methode om de efficiëntie van megakaryocyten te evalueren om proplatelets uit te breiden die in het beenmerg zijn gegroeid. Het beenmergexplantatiemodel voor muizen heeft vier belangrijke voordelen. Ten eerste zijn er geen geavanceerde technische vaardigheden vereist. Ten tweede is de tijd die nodig is om megakaryocytenverlengende voorplaatjes te verkrijgen vrij kort, slechts 6 uur voor de explantmethode, vergeleken met een minimum van 4 dagen voor een con...

De beenmergexplantatietechniek werd voor het eerst ontwikkeld door Thiéry en Bessis in 1956 om de vorming van megakaryocytencytoplasmatische extensies van ratten te beschrijven als een eerste gebeurtenis bij de vorming van bloedplaatjes1. Met behulp van fasecontrast en cinematografische technieken karakteriseerden deze auteurs de transformatie van volwassen ronde megakaryocyten in "inktvisachtige" trombocytogene cellen met cytoplasmatische extensies die dynamische bewegingen van verlenging en contractie vertonen. Deze armen worden geleidelijk dunner totdat ze volvormig worden met kleine zwellingen langs de armen en aan de uiteinden. Deze typische megakaryocytenverlengingen, verkregen in vitro en in vloeibare media, hebben bepaalde overeenkomsten met bloedplaatjes waargenomen in vast beenmerg, waar megakaryocyten lange verlengingen door de sinusoïdewandenin de bloedcirculatie uitsteken 2,3. De ontdekking en het klonen van TPO in 1994, maakte het mogelijk om megakaryocyten in cultuur te differentiëren die proplatelet-extensies kunnen vormen die lijken op die beschreven in beenmergexplantaten4,5,6. De rijping van megakaryocyten is echter veel minder efficiënt in kweekomstandigheden, met name het uitgebreide interne membraannetwerk van beenmergrijpe megakaryocyten is onderontwikkeld in gekweekte megakaryocyten, wat studies naar de mechanismen van biogenese van bloedplaatjes belemmert7,8.
We beschrijven hier het beenmergexplantatiemodel, gebaseerd op Thiéry en Bessis, om in real-time proplateletvorming van muis megakaryocyten te volgen, die volledig zijn gerijpt in hun oorspronkelijke omgeving, waardoor mogelijke in vitro artefacten en verkeerde interpretaties worden omzeild. Resultaten verkregen bij volwassen muizen van het wildtype worden gepresenteerd om het vermogen van megakaryocyten om proplatelets uit te breiden, hun morfologie en de complexiteit van proplatelets te illustreren. We introduceren ook een snelle kwantificeringsstrategie voor kwaliteitsvalidatie om de nauwkeurigheid en robuustheid van gegevens tijdens het megakaryocytenregistratieproces te garanderen. Het protocol dat hier wordt gepresenteerd, is de meest recente versie van de methode die eerder als boekhoofdstuk is gepubliceerd9.

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	<tbody>
		<tr>
			<td>5 mL syringes</td>
			<td>Terumo</td>
			<td>SS+05S1</td>
			<td></td>
		</tr>
		<tr>
			<td>21-gauge needles</td>
			<td>BD Microlance</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2.6H2O</td>
			<td>Sigma</td>
			<td>21108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwall Incubation Chambers</td>
			<td>Electron Microscopy Sciences</td>
			<td>70324-02</td>
			<td>Depth : 0,2 mm</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H-3375</td>
			<td>pH adjusted to 7.5</td>
		</tr>
		<tr>
			<td>Human serum albumin</td>
			<td>VIALEBEX</td>
			<td>authorized medication : n&#176; 3400956446995</td>
			<td>20% (200mg/mL -100mL)</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2.6H2O</td>
			<td>Sigma</td>
			<td>BVBW8448</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover Glass</td>
			<td>Electron Microscopy Sciences</td>
			<td>72200-40</td>
			<td>22 mm x 55 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica Microsystems SA, Westlar, Germany</td>
			<td>DMI8 - 514341</td>
			<td>air lens</td>
		</tr>
		<tr>
			<td>microscope camera</td>
			<td>Leica Microsystems SA, Westlar, Germany</td>
			<td>K5 CMS GmbH -14401137</td>
			<td>image resolution : 4.2 megapixel</td>
		</tr>
		<tr>
			<td>Mouse serum</td>
			<td>BioWest</td>
			<td>S2160-010</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4.H2O</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>PSG 100x</td>
			<td>Gibco, Life Technologies</td>
			<td>1037-016</td>
			<td>10,000 units/mL penicillin, 10,000 &#956;g/mL streptomycin and 29.2 mg/mL glutamine</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Electron Microscopy Sciences</td>
			<td>72000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose D (+)</td>
			<td>Sigma</td>
			<td>G8270</td>
			<td></td>
		</tr>
	</tbody>
</table>

proplatelet formation dynamics of mouse fresh bone marrow explants

De laatste fase van megakaryopoiese leidt tot cytoplasmatische uitbreidingen van volwassen megakaryocyten, de zogenaamde proplatelets. Er is veel geleerd over de proplateletvorming met behulp van in vitro-gedifferentieerde megakaryocyten; er is echter een toenemend bewijs dat conventionele kweeksystemen het differentiatie- / rijpingsproces dat plaatsvindt in het beenmerg niet getrouw samenvatten. In dit manuscript presenteren we een explantmethode die oorspronkelijk in 1956 werd beschreven door Thiéry en Bessis om megakaryocyten te visualiseren die in hun oorspronkelijke omgeving zijn gerijpt, waardoor potentiële artefacten en verkeerde interpretaties worden omzeild. Verse beenmerg wordt verzameld door de dijbenen van muizen te spoelen, in 0,5 mm doorsneden en in een incubatiekamer bij 37 °C geplaatst met een fysiologische buffer. Megakaryocyten worden geleidelijk zichtbaar aan de periferie van de explant en worden tot 6 uur waargenomen onder een omgekeerde microscoop gekoppeld aan een videocamera. Na verloop van tijd veranderen megakaryocyten van vorm, waarbij sommige cellen een bolvorm hebben en andere dikke uitbreidingen ontwikkelen of veel dunne proplatelets met uitgebreide vertakkingen uitbreiden. Er worden zowel kwalitatief als kwantitatief onderzoek verricht. Deze methode heeft het voordeel dat het eenvoudig, reproduceerbaar en snel is, omdat er talloze megakaryocyten aanwezig zijn, en klassiek vormt de helft van hen proplatelets in 6 uur vergeleken met 4 dagen voor gekweekte muizen megakaryocyten. Naast de studie van mutante muizen, is een interessante toepassing van deze methode de eenvoudige evaluatie van de farmacologische middelen op het proplatelet extensieproces, zonder het differentiatieproces dat in culturen kan optreden te verstoren.

Kwalitatieve resultaten. Aan het begin van het experiment worden alle cellen samengeperst in het beenmerggedeelte. Het duurt 30 minuten voordat de cellen duidelijk zichtbaar worden aan de periferie van de explantaten. De megakaryocyten zijn dan herkenbaar aan hun grote omvang en hun evolutie kan dan in de loop van de tijd worden bestudeerd (grootte, vorm, dynamiek, proplatelet extensie en bloedplaatjesafgifte) (Figuur 2A). Kleine megakaryocyten hebben een diameter tussen 20 ...

Watch this Scientific Journal Video about Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants at JoVE.com

Proplatelet Formation Dynamics of Mouse Fresh Bone Marrow Explants

Hier beschrijven we de beenmergexplantmethode, van monstervoorbereiding tot microscopische dia-analyse, om het vermogen van megakaryocyten te evalueren die in hun fysiologische omgeving hebben gedifferentieerd om proplatelets te vormen.

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the ...

Het algemene doel van deze procedure is om in realtime de vorming van proplatelet uit muis megakaryocyten te volgen, die in hun oorspronkelijke omgeving zijn gerijpt. Deze methode heeft het voordeel dat het eenvoudig, reproduceerbaar en snel is. Men kan veel megakaryocyten analyseren en de vorming van proplatelets in slechts zes uur visualiseren, in plaats van te wachten op de eerste dag van de kweek zoals voor muis megakaryocyten in vitro.Een interessante toepassing van deze methode is een mogelijkheid om het effect van de farmaceutische behandeling of genetische mutatie te bestuderen, met name in de stap van proplatelet extensie. Hier is er geen interferentie met het differentiatieproces, zoals in het geval van in-vitrocultuur. Deze video helpt deze belangrijke stappen van het spoelen en snijden van het beenmerg te garanderen.Er wordt ook uitgelegd hoe de morfologie en megakaryocyten moeten worden geclassificeerd, wat echt nuttig is voor de karakterisering van defecten bij trombofiele aandoeningen. Om te beginnen, verwarm de buffer van Tyrode op 37 graden Celsius en zet de verwarmingskamer van de microscoop aan om de temperatuur op 37 graden Celsius te brengen. Bereid alle benodigde gereedschappen voor, zoals een timer, incubatiekamers, vijf milliliter 21 gauge spuiten, tang, een scheermesje, Pasteur pipetten, glazen dia's en 15 milliliter centrifugebuis.Spoel het beenmerg met twee milliliter Tyrode's buffer door een naald van 21 gauge in de opening van het dijbeen aan de kniezijde te brengen en druk langzaam op de zuiger om een intacte mergcilinder te halen. Gebruik een plastic pipet van drie minuten om het intacte beenmerg voorzichtig en voorzichtig op een glazen dia over te brengen. Snijd de uiteinden van het merg af die mogelijk zijn samengedrukt op het moment van de spoeling.Snijd vervolgens dunne transversale secties van 0,5 millimeter dikte met behulp van een scherp scheermesje om beschadiging van de megakaryocyten te voorkomen. Verzamel met behulp van een plastic pipet 10 secties in een buis van één milliliter met de buffer van Tyrode. Breng de secties voorzichtig over naar een incubatiekamer met een diameter van 13 millimeter.Zuig de buffer op en stel het volume in op 30 microliter van Tyrode's buffer aangevuld met 5%muizenserum. Plaats de secties op een afstand. Sluit de zelfklevende kamer af met een afdekslip van 22 bij 55 millimeter, waarbij de afdekslip wordt hellend om de vorming van luchtbellen te voorkomen.Plaats de kamer in de verwarmingskamer bij 37 graden Celsius. Start de chronometer en voer het experiment zes uur lang uit bij 37 graden Celsius, maak video's om de transformatie van de megakaryocyten vast te leggen. Na 30 minuten migreren de mergcellen geleidelijk naar de periferie van de explant en vormen ze een monolaag.Na een uur incubatie kunnen megakaryocyten worden geïdentificeerd aan de hand van hun grote omvang en polylobulated kernen. Het aantal megakaryocyten neemt toe na drie uur incubatie en sommige hebben lange verlengingen. Teken een kaart om elke sectie in de incubatiekamer te lokaliseren.Identificeer na een uur de zichtbare megakaryocyten, die gigantische polylobulated cellen zijn aan de periferie van elke sectie, en plot hun posities op de tekening. Herhaal deze procedure na drie en zes uur. Megakaryocyten worden handmatig geteld en geclassificeerd volgens hun morfologie op drie en zes uur na het afdichten van de incubatiekamer.Ze zijn geclassificeerd als klein, groot met dikke intentie en proplatelet uitschuivend. Met behulp van mapping kan hun evolutie in de loop van de tijd worden gevolgd. De resultaten worden uitgedrukt als een percentage van elke klasse op elk observatietijds tijdstip.Klassiek strekt de helft van de megakaryocyten die zichtbaar zijn aan de periferie proplatelets uit na zes uur voor wild type muisbeenmerg. Het lot van ronde megakaryocyten werd gevolgd door het vastleggen van sequentiële beelden in de loop van de tijd om in beeld te krijgen hoe ze proplatelets vormen. Een belangrijk ding om te onthouden bij het proberen van deze procedure is om de explants op 37 graden Celsius te houden voor de duur van het experiment.Een andere temperatuur kan de resultaten veranderen. Interessant is dat het explant-protocol kan worden gebruikt na introverte microscopie-experimenten. Uiteindelijk zullen de megakaryocyten in de beenmergexplantaten van nature fluorescerend zijn en kan hun gedrag gemakkelijk worden onderzocht.Dit maakt het mogelijk om verschillende behandelingen op dezelfde dieren te combineren en zo het aantal muizen te verminderen. Kortom, de explantmethode is snel, eenvoudig en biedt veel informatie over de capaciteit van natuurlijke megakaryocyten aan externe proplatelets. De resulterende kwalitatieve en kwantitatieve bevinding zijn de sleutel tot ons begrip van trombofiele ziekte.in een fysiologische of pathologische context.

proplatelet-formation-dynamics-of-mouse-fresh-bone-marrow-explants

Research

JoVE Journal

Biology

Culture of myeloid dendritic cells from bone marrow precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to infection. Because these are the most potent antigen presenting cells, DCs are being increasingly used as a vaccine vector to study the induction of antigen-specific immune responses. In this video, we demonstrate the procedure for harvesting tibias and femurs from a donor mouse, processing the bone marrow and differentiating DCs in vitro. The properties of DCs change following stimulation: immature dendritic cells are potent phagocytes, whereas mature DCs are capable of antigen presentation and interaction with CD4+ and CD8+ T cells. This change in functional activity corresponds with the upregulation of cell surface markers and cytokine production. Many agents can be used to mature DCs, including cytokines and toll-like receptor ligands. In this video, we demonstrate flow cytometric comparisons of expression of two co-stimulatory molecules, CD86 and CD40, and the cytokine, IL-12, following overnight stimulation with CpG or mock treatment. After differentiation, DCs can be further manipulated for use as a vaccine vector or to generate antigen-specific immune responses by in vitro pulsing using peptides or proteins, or transduced using recombinant viral vectors. 

This video demonstrates the procedure for differentiating myeloid dendritic cells from mouse bone marrow. Isolation of mouse tibia and femur, and processing of bone marrow are demonstrated. Pictures demonstrating cell morphology before and after differentiation, and figures depicting cell phenotype and IL-12 production following maturation using CpG are shown.

Cultuur van myeloïde dendritische cellen uit beenmerg precursors

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Generatie van beenmerg afgeleide Murine dendritische cellen voor gebruik in twee-foton Imaging

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

De vervaardiging van primaire hematopoietische cellen culturen uit Murine Bone Marrow voor Elektroporatie

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Sturen van hematopoietische cellen naar het beenmerg

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo 4-dimensionale Tracking van hematopoietische stamcellen en progenitorcellen in volwassen muis calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

Osteoclast Afleiding van Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

Trans-inner Cell Mass injectie van embryonale stamcellen tot hogere Chimerism tarieven leidt

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Expression of Fluorescent Fusion Proteins in Murine Bone Marrow-derived Dendritic Cells and Macrophages

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation of an adaptive immune response. Experimental work with these cells is rather challenging. Their abundance in organs and tissues is relatively low. As a result, they cannot be isolated in large numbers. They are also difficult to transfect with cDNA constructs. In the murine model, these problems can be partially overcome by in vitro differentiation from bone marrow progenitors in the presence of M-CSF for macrophages or GM-CSF for dendritic cells. In this way, it is possible to obtain large amounts of these cells from very few animals. Moreover, bone marrow progenitors can be transduced with retroviral vectors carrying cDNA constructs during early stages of cultivation prior to their differentiation into bone marrow derived dendritic cells and macrophages. Thus, retroviral transduction followed by differentiation in vitro can be used to express various cDNA constructs in these cells. The ability to express ectopic proteins substantially extends the range of experiments that can be performed on these cells, including live cell imaging of fluorescent proteins, tandem purifications for interactome analyses, structure-function analyses, monitoring of cellular functions with biosensors and many others. In this article, we describe a detailed protocol for retroviral transduction of murine bone marrow derived dendritic cells and macrophages with vectors coding for fluorescently-tagged proteins. On the example of two adaptor proteins, OPAL1 and PSTPIP2, we demonstrate its practical application in flow cytometry and microscopy. We also discuss the advantages and limitations of this approach.

In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.

Expressie van fluorescerende Fusion eiwitten in lymfkliertest beenmerg-afgeleide dendritische cellen en macrofagen

Dendritic cells and macrophages are crucial cells that form the first line of defense against pathogens. They also play important roles in the initiation ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Isolatie, zuivering en differentiatie van osteoclast precursoren van het Rat beenmerg

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

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

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

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

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

In vivo Twee-foton Beeldvorming van Megakaryocyten en Proplatelets in de Muis Schedel Beenmerg

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