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

Forfatterne ønsker å takke Jean-Yves Rinckel, Julie Boscher, Patricia Laeuffer, Monique Freund, Ketty Knez-Hippert for teknisk assistanse. Dette arbeidet har blitt støttet av ANR (Agence National de la Recherche) Grant ANR-17-CE14-0001-01 og ANR-18-CE14-0037.

Alle dyreforsøk ble utført i samsvar med europeiske standarder 2010/63/EU og CREMEAS-komiteen for etikk for dyreforsøk ved Universitetet i Strasbourg (Comité Régional d'Ethique en Matière d'Expérimentation Animale Strasbourg).
1. Utarbeidelse av reagenser
<ol><li>Forbered reagenser som beskrevet i tabell 1.<ol>
<li>For lager I, oppløs hvert pulver separat. Forsikre deg om at osmolariteten til preparatet er høyere enn 295 mOsm / L. Denne løsningen kan lagres ved 4 ?...

<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>

Her beskriver vi en enkel og rimelig in vitro-metode for å evaluere effektiviteten til megakaryocytter for å utvide proplater som har vokst i benmargen. Benmargens utvisningsmodell for mus har fire hovedfordeler. For det første er det ingen avanserte tekniske ferdigheter som kreves. For det andre er tiden som trengs for å oppnå megakaryocyttforlengende proplatelets ganske kort, bare 6 timer for explant-metoden, sammenlignet med minst 4 dager for en konvensjonell kulturmetode som starter fra museforfedre. Fo...

Benmargsutvisningsteknikken ble først utviklet av Thiéry og Bessis i 1956 for å beskrive dannelsen av rotte megakaryocytt cytoplasmatiske utvidelser som en innledende hendelse i blodplateformasjon1. Ved hjelp av fasekontrast og kinematografiske teknikker karakteriserte disse forfatterne transformasjonen av modne runde megakaryocytter til "blekksprutlignende" trombocytogene celler med cytoplasmatiske utvidelser som viser dynamiske bevegelser av forlengelse og sammentrekning. Disse armene blir gradvis tynnere til de blir filiform med små hevelser langs armene og på spissene. Disse typiske megakaryocyttforlengelsene, oppnådd in vitro og i flytende medier, har visse likheter med blodplater observert i fast benmarg, hvor megakaryocytter stikker lange forlengelser gjennom bihuleveggene inn i blodsirkulasjonen2,3. Oppdagelsen og kloningen av TPO i 1994, fikk lov til å skille megakaryocytter i kultur i stand til å danne proplatelet utvidelser som ligner de som er beskrevet i benmarg explants4,5,6. Imidlertid er megakaryocyttmodning langt mindre effektiv i kulturforhold, spesielt det omfattende indre membrannettverket av benmarg modnet megakaryocytter er underutviklet i dyrkede megakaryocytter, noe som hemmer studier på mekanismene for blodplatebiogenese7,8.
Vi detaljerer her benmargen explant modell, basert på Thiéry og Bessis, å følge i sanntid proplatelet dannelse av mus megakaryocytter, som har fullstendig modnet i sitt opprinnelige miljø, og dermed omgå mulige in vitro artefakter og feiltolkninger. Resultater oppnådd i villtype voksne mus presenteres for å illustrere megakaryocyttens evne til å utvide proplater, deres morfologi og kompleksiteten til proplatelets. Vi introduserer også en rask kvantifiseringsstrategi for kvalitetsvalidering for å sikre datanøyaktighet og robusthet under megakaryocyttopptaksprosessen. Protokollen som presenteres her, er den nyeste versjonen av metoden som ble publisert som et bokkapittel tidligere9.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

Den siste fasen av megakaryopoiesis fører til cytoplasmatiske utvidelser fra modne megakaryocytter, de såkalte proplatelets. Mye har blitt lært om proplateletformasjonen ved hjelp av in vitro-differensierte megakaryocytter; Det er imidlertid et økende bevis på at konvensjonelle kultursystemer ikke trofast rekapitulerer differensierings- / modningsprosessen som foregår inne i benmargen. I dette manuskriptet presenterer vi en explant-metode som opprinnelig ble beskrevet i 1956 av Thiéry og Bessis for å visualisere megakaryocytter som har modnet i sitt opprinnelige miljø, og dermed omgå potensielle gjenstander og feiltolkninger. Friske benmarger samles ved å skylle lårbenene til mus, skivet i 0,5 mm tverrsnitt, og plasseres i et inkubasjonskammer ved 37 °C som inneholder en fysiologisk buffer. Megakaryocytter blir gradvis synlige i den utplantede periferien og observeres opptil 6 timer under et invertert mikroskop koblet til et videokamera. Over tid endrer megakaryocytter sin form, med noen celler som har en sfærisk form og andre utvikler tykke utvidelser eller utvider mange tynne proplatelets med omfattende forgrening. Både kvalitative og kvantitative undersøkelser utføres. Denne metoden har fordelen av å være enkel, reproduserbar og rask som mange megakaryocytter er til stede, og klassisk halvparten av dem danner proplatelets på 6 timer sammenlignet med 4 dager for dyrkede mus megakaryocytter. I tillegg til studiet av mutante mus er en interessant anvendelse av denne metoden den enkle evalueringen av farmakologiske midler på proplateletforlengelsesprosessen, uten å forstyrre differensieringsprosessen som kan oppstå i kulturer.

Kvalitative resultater. I begynnelsen av eksperimentet komprimeres alle celler i benmargsdelen. Det tar 30 min for cellene å bli tydelig synlige i periferien av explants. Megakaryocyttene er da gjenkjennelige av sin store størrelse, og deres evolusjon kan deretter studeres over tid (størrelse, form, dynamisk, proplateletforlengelse og blodplateutløsning) (Figur 2A). Små megakaryocytter har en diameter mellom 20 og 30 μm og deres kjerner er polylobulert mens modne runde...

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

Her beskriver vi benmargseksekale metoder, fra prøvepreparering til mikroskopisk lysbildeanalyse, for å evaluere evnen til megakaryocytter som har differensiert i deres fysiologiske miljø til å danne proplatelets.

Proplatelet formasjonsdynamikk av mus frisk benmarg explants

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

Det overordnede målet med denne prosedyren er å følge i sanntid dannelsen av proplatelet fra musen megakaryocytter, som modnet i sitt opprinnelige miljø. Denne metoden har fordelen av å være enkel, reproduserbar og rask. Man kan analysere mange megakaryocytter og visualisere dannelsen av proplatelets på bare seks timer, i stedet for å vente på den første kulturdagen som for mus megakaryocytter in vitro.En interessant anvendelse av denne metoden er en mulighet til å studere effekten av farmasøytisk behandling eller genetisk mutasjon, spesielt i trinnet for proplatelet forlengelse. Her er det ingen forstyrrelser i differensieringsprosessen, som i tilfelle in-vitro kultur. Denne videoen bidrar til å sikre disse viktige trinnene for spyling og kutting av benmargen.Det forklares også hvordan man klassifiserer morfologi og megakaryocytter, noe som er veldig nyttig for karakterisering av defekter ved trombofil sykdom. Til å begynne med, varm Tyrodes buffer ved 37 grader Celsius, og slå på mikroskopets varmekammer for å bringe temperaturen til 37 grader Celsius. Forbered alle nødvendige verktøy, for eksempel en timer, inkubasjonskamre, fem milliliter 21 gauge sprøyter, tang, et barberblad, Pasteur pipetter, glasssklier og 15 milliliter sentrifugerør.Skyll benmargen med to milliliter Tyrodes buffer ved å introdusere en 21 gauge nål i åpningen av lårbenet på knesiden, og trykk stempelet sakte for å hente en intakt margsylinder. Bruk en tre-minutters liter plastpipette for å forsiktig og forsiktig overføre den intakte benmargen til en glasssklie. Klipp av endene av margen som kan ha blitt komprimert på tidspunktet for spylingen.Klipp deretter tynne tverrgående seksjoner på 0,5 millimeter tykkelse ved hjelp av et skarpt barberblad for å unngå å skade megakaryocyttene. Bruk en plastpipette til å samle 10 seksjoner i et milliliterrør som inneholder Tyrodes buffer. Overfør forsiktig seksjonene til et inkubasjonskammer med en diameter på 13 millimeter.Aspirer bufferen og juster volumet til 30 mikrolitere av Tyrodes buffer supplert med 5 % museserum. Plasser seksjonene på avstand. Forsegle det selvklebende kammeret med en 22 x 55 millimeter dekselglidning, og hell dekselet for å unngå dannelse av luftbobler.Plasser kammeret i varmekammeret ved 37 grader Celsius. Start kronometeret og kjør eksperimentet i seks timer ved 37 grader Celsius, Lag videoer for å spille inn transformasjonen av megakaryocyttene. Etter 30 minutter migrerer margcellene gradvis til periferien av explant som danner et monolag.Etter en time med inkubasjon kan megakaryocytter identifiseres av deres store størrelse og polylobuerte kjerner. Antall megakaryocytter øker etter tre timer med inkubasjon, og noen har lange utvidelser. Tegn et kart for å lokalisere hver seksjon i inkubasjonskammeret.Etter en time identifiserer du de synlige megakaryocyttene, som er gigantiske polylobuerte celler på hver seksjons periferi, og plotter sine posisjoner på tegningen. Gjenta denne prosedyren etter tre og seks timer. Megakaryocytter telles manuelt og klassifiseres i henhold til deres morfologi på tre og seks timer etter forsegling av inkubasjonskammeret.De er klassifisert som små, store med tykk intensjon og proplatelet som strekker seg. Ved hjelp av kartlegging kan deres evolusjon følges over tid. Resultatene uttrykkes som en prosentandel av hver klasse på hvert observasjonstidspunkt.Klassisk utvider halvparten av megakaryocyttene som er synlige i periferien proplatelets på seks timer for vill type musebenmarg. Skjebnen til runde megakaryocytter ble etterfulgt av å fange sekvensielle bilder over tid for å bilde hvordan de danner proplatelets. En viktig ting å huske når du prøver denne prosedyren er å holde utløpene på 37 grader Celsius i løpet av eksperimentet.En annen temperatur kan endre resultatene. Interessant nok kan explantprotokollen brukes etter introverte mikroskopieksperimenter. Til slutt vil megakaryocyttene i benmargsutløpene være naturlig fluorescerende, og deres oppførsel kan lett undersøkes.Dette gjør det mulig å kombinere forskjellig behandling på de samme dyrene og dermed redusere antall mus. Til slutt er explant-metoden rask, enkel og gir mange opplysninger om kapasiteten til naturlige megakaryocytter til eksterne proplater. Det resulterende kvalitative og kvantitative funnet er nøkkelen til vår forståelse av trombofil sykdom.i en fysiologisk eller patologisk kontekst.

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.

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.

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.

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 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 Sporing av Hematopoietiske Stem og stamceller i Adult Mouse 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 Utledning fra 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-indre cellen masse injeksjon av embryonale stamceller fører til høyere Chimerism priser

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.

Uttrykk for fluorescerende Fusion proteiner i Murine Ben margtransplantasjon-avledet dendrittiske celler og 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.

Isolasjon, rensing, og differensiering av Osteoclast forløpere fra Rat Bone marg

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 To-foton bildebehandling av Megakaryocytes og Proplatelets i Mus 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 ...