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

Författarna vill tacka Jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert för tekniskt stöd. Detta arbete har fått stöd av ANR (Agence National de la Recherche) Grant ANR-17-CE14-0001-01 och ANR-18-CE14-0037.

Alla djurförsök utfördes i enlighet med europeiska standarder 2010/63/EU och CREMEAS Committee on the Ethics of Animal Experiments vid universitetet i Strasbourg (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg).
1. Beredning av reagenser
<ol><li>Förbered reagenser enligt beskrivningen i tabell 1.<ol>
<li>Lös upp varje pulver separat för buljongen I. Se till att preparatets osmolaritet är högre än 295 mOsm/L. Denna lösning kan förvara...

<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>

Här beskriver vi en enkel och billig in vitro-metod för att utvärdera effektiviteten hos megakaryocyter för att utöka proplatelets som har vuxit i benmärgen. Benmärg explant modellen för musen har fyra huvudsakliga fördelar. För det första krävs inga avancerade tekniska färdigheter. För det andra är den tid som behövs för att erhålla megakaryocytförlängande proplatelets ganska kort, bara 6 timmar för explantmetoden, jämfört med minst 4 dagar för en konventionell odlingsmetod som börjar f...

Benmärg explant tekniken utvecklades först av Thiéry och Bessis 1956 för att beskriva bildandet av råtta megakaryocyt cytoplasmic förlängningar som en första händelse i trombocyt bildandet1. Med hjälp av faskontrast och cinematographic tekniker karakteriserade dessa författare omvandlingen av mogna runda megakaryocyter till "bläckfiskliknande" trombocytogena celler med cytoplasmiska förlängningar som visar dynamiska rörelser av förlängning och sammandragning. Dessa armar blir gradvis tunnare tills de blir filiform med små svullnader längs armarna och vid spetsarna. Dessa typiska megakaryocytförlängningar, erhållna in vitro och i flytande medier, har vissa likheter med trombocyter som observerats i fast benmärg, där megakaryocyter sticker ut långa förlängningar genom sinusoidväggarna in i blodcirkulationen2,3. Upptäckten och kloningen av TPO 1994, tillåtet att skilja megakaryocyter i kultur som kan bilda proplatelet förlängningar som liknar de som beskrivs i benmärg explanter4,5,6. Megakaryocytmamognad är dock mycket mindre effektiv under odlingsförhållanden, särskilt det omfattande inre membrannätverket av benmärgsmogna megakaryocyter är underutvecklat i odlade megakaryocyter, vilket hindrar studier om mekanismerna för trombocytbiogenes7,8.
Vi beskriver här benmärg explant modellen, baserat på Thiéry och Bessis, att följa i realtid proplatelet bildandet av mus megakaryocyter, som har full mognad i sin inhemska miljö, vilket kringgår möjliga in vitro artefakter och feltolkningar. Resultat erhållna i vilda vuxna möss presenteras för att illustrera megakaryocytens förmåga att förlänga proplatelets, deras morfologi och komplexiteten hos proplatelets. Vi introducerar också en snabb kvantifieringsstrategi för kvalitetsvalidering för att säkerställa datanoggrannhet och robusthet under megakaryocytinspelningsprocessen. Protokollet som presenteras här är den senaste versionen av metoden som publicerades som ett bokkapitel tidigare9.

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

Det sista steget i megakaryopoiesis leder till cytoplasmiska förlängningar från mogna megakaryocyter, de så kallade proplateletsna. Mycket har lärts om proplatelet bildas med hjälp av in vitro-differentierademegakaryocyter; Det finns dock allt fler bevis för att konventionella odlingssystem inte troget rekapitulerar differentierings-/mognadsprocessen som sker inuti benmärgen. I detta manuskript presenterar vi en explantmetod som ursprungligen beskrevs 1956 av Thiéry och Bessis för att visualisera megakaryocyter som har mognat i sin ursprungliga miljö, vilket kringgår potentiella artefakter och feltolkningar. Färska benmärgar samlas in genom att spola lårbenen hos möss, skivas i 0,5 mm tvärsnitt och placeras i en inkubationskammare vid 37 °C som innehåller en fysiologisk buffert. Megakaryocyter blir gradvis synliga vid explantens periferi och observeras upp till 6 timmar under ett inverterat mikroskop kopplat till en videokamera. Med tiden ändrar megakaryocyter sin form, med vissa celler som har en sfärisk form och andra utvecklar tjocka förlängningar eller utökar många tunna proplatelets med omfattande förgrening. Både kvalitativa och kvantitativa undersökningar genomförs. Denna metod har fördelen att vara enkel, reproducerbar och snabb som många megakaryocyter är närvarande, och klassiskt hälften av dem bildar proplatelets i 6 timmar jämfört med 4 dagar för odlade mus megakaryocyter. Förutom studien av muterade möss är en intressant tillämpning av denna metod den enkla utvärderingen av farmakologiska medel på proplatelet förlängningsprocessen, utan att störa differentieringsprocessen som kan uppstå i kulturer.

Kvalitativa resultat. I början av experimentet komprimeras alla celler i benmärgssektionen. Det tar 30 minuter för cellerna att bli tydligt synliga i explanternas periferi. Megakaryocyterna känns sedan igen av sin stora storlek och deras utveckling kan sedan studeras med tiden (storlek, form, dynamik, proplateletförlängning och trombocytfrisättning) (figur 2A). Små megakaryocyter har en diameter mellan 20 och 30 μm och deras kärnor polylobuleras medan mogna runda m...

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

Här beskriver vi benmärg explant metoden, från prov beredning till mikroskopisk glidanalys, att utvärdera förmågan hos megakaryocyter som har differentierat i sin fysiologiska miljö att bilda proplatelets.

Proplatelet Formation Dynamics av mus färska benmärg explanter

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

Det övergripande målet med detta förfarande är att i realtid följa bildandet av proplatelet från mus megakaryocyter, som mognade i sin ursprungliga miljö. Denna metod har fördelen att vara enkel, reproducerbar och snabb. Man kan analysera många megakaryocyter och visualisera bildandet av proplatelets på bara sex timmar, istället för att vänta på den första kulturdagen som för mus megakaryocyter in vitro.En intressant tillämpning av denna metod är en möjlighet att studera effekten av läkemedelsbehandling eller genetisk mutation, särskilt i steget av proplatelet förlängning. Här finns det ingen inblandning i differentieringsprocessen, som i fallet med in vitro-kulturen. Denna video hjälper till att säkerställa dessa viktiga steg för att spola och skära benmärgen.Det förklaras också hur man klassificerar morfologi och megakaryocyter, vilket verkligen är användbart för karakterisering av defekter vid trombofil sjukdom. Till att börja med värm Tyrlids buffert vid 37 grader Celsius och slå på mikroskopets värmekammare för att få temperaturen till 37 grader Celsius. Förbered alla nödvändiga verktyg, såsom en timer, inkubationskammare, fem milliliter 21 gauge sprutor, tång, ett rakblad, Pasteur pipetter, glasrutschbanor och 15 ml centrifugrör.Spola benmärgen med två milliliter tyrodes buffert genom att föra in en 21 gauge nål i lårbenets öppning på knäsidan och tryck långsamt på kolven för att hämta en intakt märgcylinder. Använd en tre minuters plastpipett för att försiktigt och försiktigt överföra den intakta benmärgen till en glasrutschbana. Skär av ändarna av märgen som kan ha komprimerats vid tidpunkten för spolningen.Skär sedan tunna tvärgående sektioner med 0,5 millimeters tjocklek med hjälp av ett vasst rakblad för att undvika att skada megakaryocyterna. Samla 10 sektioner i ett en milliliterrör som innehåller Tyrodes buffert med hjälp av en plastpipetter. Överför försiktigt sektionerna till en inkubationskammare med en diameter på 13 millimeter.Aspirera bufferten och justera volymen till 30 mikroliter av Tyredes buffert kompletterad med 5% musserum. Placera sektionerna på avstånd. Försegla den självhäftande kammaren med en 22 x 55 millimeters täckglidning, luta locket glida för att undvika bildandet av luftbubblor.Placera kammaren i värmekammaren vid 37 grader Celsius. Starta kronometern och kör experimentet i sex timmar vid 37 grader Celsius, Gör videor för att spela in omvandlingen av megakaryocyterna. Efter 30 minuter migrerar märgcellerna gradvis till periferin av explanten som bildar ett monoskikt.Efter en timmes inkubation kan megakaryocyter identifieras av deras stora storlek och polylobulated atomkärnor. Antalet megakaryocyter ökar efter tre timmars inkubation, och vissa har långa förlängningar. Rita en karta för att lokalisera varje sektion i inkubationskammaren.Efter en timme identifierar du de synliga megakaryocyterna, som är gigantiska polylobulerade celler i varje sektions periferi, och ritar sina positioner på ritningen. Upprepa denna procedur efter tre och sex timmar. Megakaryocyter räknas manuellt och klassificeras enligt deras morfologi vid tre och sex timmar efter tätning av inkubationskammaren.De klassificeras som små, stora med tjock avsikt och proplatelet som sträcker sig. Med hjälp av kartläggning kan deras utveckling följas över tid. Resultaten uttrycks i procent av varje klass vid varje observationstid.Klassiskt förlänger hälften av de megakaryocyter som är synliga i periferin proplatelets vid sex timmar för vild typ mus benmärg. Ödet för runda megakaryocyter följdes av att fånga sekventiella bilder över tid för att avbilda hur de bildar proplatelets. En viktig sak att komma ihåg när du försöker denna procedur är att hålla explanterna vid 37 grader Celsius under experimentets varaktighet.En annan temperatur kan ändra resultaten. Intressant nog kan explant-protokollet användas efter introverta mikroskopiexperiment. I slutändan kommer megakaryocyterna i benmärgsexplanterna att vara naturligt fluorescerande, och deras beteende kan lätt undersökas.Detta gör det möjligt att kombinera olika behandling på samma djur och därmed minska antalet möss. Sammanfattningsvis är explantmetoden snabb, enkel och ger många information om kapaciteten hos naturliga megakaryocyter till externa proplatelets. Den resulterande kvalitativa och kvantitativa konstaterandet är nyckeln till vår förståelse av trombofila sjukdom.i ett fysiologiskt eller patologiskt sammanhang.

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.

Kultur av myeloisk dendritiska celler från benmärg prekursorer

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.

Generering av benmärg härledda Murine dendritiska celler för användning i 2-fotonen 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.

Beredningen av primära hematopoietisk cell kulturer från Murine Bone Marrow för elektroporation

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.

Homing av hematopoetiska celler i benmärgen

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-Dimensional Spårning av Hematopoietic Stem och stamceller i vuxen mus calvarial Benmärg

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.

Osteoklast Härledning från benmärg från mus

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 injektion av embryonala stamceller leder till högre chimärism

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.

Uttryck för fluorescerande fusionsproteinerna i murina benmärg-derived dendritiska celler och makrofager

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.

Isolering, rening och differentiering av Osteoklast prekursorer från råtta benmärg

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 Två-foton imaging av Megakaryocytes och Proplatelets i musskalle benmärgen

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