Name: stimulation of notch signaling in mouse osteoclast precursors
Uploaded: 2017-02-28T15:00:00
Description: Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

This work was supported by the University of Pennsylvania Center for Musculoskeletal Disorders (5 P30 AR050950-09), a grant from the AO Foundation (S-16-12A), the Philadelphia VA Medical Center Translational Musculoskeletal Research Center, and an intramural orthopaedic surgery departmental research development fund. JWA is supported by the University of Pennsylvania Postdoctoral Opportunities in Research and Teaching (PENN-PORT) fellowship funded by the National Institute of General Medical Sciences Institutional Research and Career Development Award (IRACDA; 5 K12 GM081259-08).

Toute recherche impliquant des animaux vertébrés a été réalisée conformément aux protocoles approuvés par l&#39;Université de Pennsylvanie Institutional Animal Care et utilisation Commission (IACUC). 1. Culture Media Préparation <ol><li> Préparer α-MEM par dissolution de milieu (MEM) en poudre essentiel minimum et 1,9 g de bicarbonate de sodium dans 900 ml d&#39; H 2 O et compléter avec 2 mM de L-glutamine, 100 unités / ml de pénicilline, 100 pg / ml de streptomyc...

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D., Shimada, I. S., Sherwin, C., Spees, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch1-STAT3-ETBR+signaling+axis+controls+reactive+astrocyte+proliferation+after+brain+injury.">Notch1-STAT3-ETBR signaling axis controls reactive astrocyte proliferation after brain injury.</a> Proc Natl Acad Sci U S A. 112 (28), 8726-8731 (2015).</li><li>Kostianovsky, A. M., Maier, L. M., Baecher-Allan, C., Anderson, A. C., Anderson, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+gene+related+to+anergy+in+lymphocytes+is+associated+with+Notch-mediated+human+T+cell+suppression.">Up-regulation of gene related to anergy in lymphocytes is associated with Notch-mediated human T cell suppression.</a> J Immunol. 178 (10), 6158-6163 (2007).</li><li>Teitelbaum, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoclasts:+what+do+they+do+and+how+do+they+do+it?.">Osteoclasts: what do they do and how do they do it?.</a> Am J Pathol. 170 (2), 427-435 (2007).</li><li>Ma, J., 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=Disruption+of+the+transcription+factor+RBP-J+results+in+osteopenia+attributable+to+attenuated+osteoclast+differentiation.">Disruption of the transcription factor RBP-J results in osteopenia attributable to attenuated osteoclast differentiation.</a> Mol Biol Rep. 40 (3), 2097-2105 (2013).</li><li>Bai, 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=NOTCH1+regulates+osteoclastogenesis+directly+in+osteoclast+precursors+and+indirectly+via+osteoblast+lineage+cells.">NOTCH1 regulates osteoclastogenesis directly in osteoclast precursors and indirectly via osteoblast lineage cells.</a> J Biol Chem. 283 (10), 6509-6518 (2008).</li><li>Yamada, 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=Regulation+of+osteoclast+development+by+Notch+signaling+directed+to+osteoclast+precursors+and+through+stromal+cells.">Regulation of osteoclast development by Notch signaling directed to osteoclast precursors and through stromal cells.</a> Blood. 101 (6), 2227-2234 (2003).</li><li>Sethi, N., Dai, X., Winter, C. G., Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-derived+JAGGED1+promotes+osteolytic+bone+metastasis+of+breast+cancer+by+engaging+notch+signaling+in+bone+cells.">Tumor-derived JAGGED1 promotes osteolytic bone metastasis of breast cancer by engaging notch signaling in bone cells.</a> Cancer Cell. 19 (2), 192-205 (2011).</li><li>Sekine, 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=Differential+regulation+of+osteoclastogenesis+by+Notch2/Delta-like+1+and+Notch1/Jagged1+axes.">Differential regulation of osteoclastogenesis by Notch2/Delta-like 1 and Notch1/Jagged1 axes.</a> Arthritis Res Ther. 14 (2), R45 (2012).</li><li>Ashley, J. W., Ahn, J., Hankenson, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+Signaling+Promotes+Osteoclast+Maturation+and+Resorptive+Activity.">Notch Signaling Promotes Osteoclast Maturation and Resorptive Activity.</a> J Cell Biochem. 116 (11), 2598-2609 (2015).</li><li>Fukushima, H., 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=The+association+of+Notch2+and+NF-kappaB+accelerates+RANKL-induced+osteoclastogenesis.">The association of Notch2 and NF-kappaB accelerates RANKL-induced osteoclastogenesis.</a> Mol Cell Biol. 28 (20), 6402-6412 (2008).</li><li>Canalis, E., Schilling, L., Yee, S. P., Lee, S. 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Les étapes critiques au sein du protocole Culture et dans la différenciation in vitro de précurseurs d&#39;ostéoclastes fournit une plate - forme utile pour l&#39; étude des mécanismes moléculaires de l&#39; ostéoclastogenèse et l&#39; identification de cibles thérapeutiques pour la régénération osseuse et la préservation de la masse osseuse. Lorsque la culture de précurseurs d&#39;ostéoclastes de souris, l&#39;élément le plus critique est l&#39;entretien des...

La voie de signalisation Notch mammifère est homologue à la même voie dans Drosophila melanogaster et se compose de quatre récepteurs transmembranaire de Notch (Notch1-4) et cinq ligands liés à la membrane du Jagged (JAG1 &amp; JAG2) et Delta-like (DLL1, 3, &amp; 4) familles 1. Signalisation Notch est déclenchée lorsque le récepteur Notch dans une cellule réceptrice est liée par un ligand sur une cellule de transmission 2. Au cours de cette trans-activation, le ligand Notch lié à la membrane produit une force d&#39; étirement sur le récepteur Notch liée à la membrane 3, 4. La force d&#39;étirement de la liaison du ligand induit des changements de conformation dans le récepteur Notch qui facilitent le clivage extracellulaire du récepteur de TNF-alpha enzyme de conversion (TACE), suivie d&#39;un événement de clivage intracellulaire médiée par une gamma sécrétase complexe (γ-sécrétase) contenant préséniline. γ-secrétase libère l&#39;int Notchdomaine racellular (NICD) qui translocation dans le noyau où il forme un complexe d&#39;activation transcriptionnelle avec CBF-1-Su (H) -Lag-1 (CSL), Mastermind-like (GAML), et les facteurs spécifiques de type cellulaire pour conduire l&#39;expression de cibler des gènes 5. Les éléments mécaniques du résultat de l&#39; activation de signalisation Notch dans le besoin de procédés uniques d&#39;activation de la voie Notch in vitro. ligands de Notch solubles peuvent se lier à Notch récepteurs, mais ne parviennent pas à produire des forces d&#39;étirement nécessaires à la libération NICD tout en même temps inhibition compétitive de liaison des ligands de Notch associés aux cellules. Ainsi, l&#39; addition de ligands de Notch solubles dans le milieu de culture peut atténuer Notch normale de signalisation 6, 7. Heureusement, les ligands de Notch peuvent induire une libération NICD si elles sont fixées à un substrat rigide approprié 5, 8, 9,10. Ensemencer des cellules sur des substrats de culture revêtues de ligand ou de l&#39;application des billes de ligand revêtu à des cellules peut aussi activer la signalisation Notch, et le choix entre les deux dépend essentiellement du temps désiré de stimulation de l&#39;entaille. Pour une activation immédiate, temporaire signalisation Notch, comme cela serait souhaité au cours du point médian d&#39;une analyse fonctionnelle ou de la différenciation, le ligand de Notch peut être lié à des billes d&#39;agarose, appliqué à des cellules en culture, et on le lave à tout moment. Pour en savoir plus soutenue signalisation Notch à partir du début d&#39;une période de culture, des plaques de culture tissulaire peuvent être enrobés avec le ligand avant l&#39;ensemencement des cellules. Aux fins du présent protocole, des procédés sont réalisés en utilisant des précurseurs d&#39;ostéoclastes de souris, mais les méthodes et variations des procédés décrits ici sont applicables à une grande variété de types de cellules 6, 11, 12, 13, </sup&gt; 14. Les ostéoclastes sont des cellules terminalement différenciées lignage hématopoiétiques qui sont responsables de la résorption du tissu osseux, et ils sont impliqués dans de multiples troubles de la perte osseuse 15. Ainsi, dans l&#39; étude in vitro de la différenciation des ostéoclastes à partir de leurs précurseurs monocytes / macrophages-lignage et les mécanismes moléculaires contrôlant leur fonction est essentielle à une meilleure compréhension des ostéoclastes et le développement de nouvelles thérapies osseuses régénérant. Alors qu&#39;il est maintenant généralement admis que la signalisation Notch joue un rôle positif dans la différenciation et la fonction des ostéoclastes, les variations dans les deux Notch signalisation stimulation et ostéoclastes culture précurseur et la différenciation ont conduit à des résultats contradictoires initialement 16, 17, 18, 19. Un examen plus approfondi des différences dans les méthodes et l&#39;utilisation des ressources génétiquesmodèles ont grandement clarifié le rôle de la signalisation Notch dans ostéoclastogenèse, mais l&#39; application des méthodes de stimulation et de culture Notch standardisés pourraient empêcher de telles controverses dans les études futures de signalisation Notch dans d&#39; autres types de cellules 20, 21, 22, 23. Il existe plusieurs méthodes pour la culture et la différenciation des précurseurs d&#39;ostéoclastes de souris, et, comme avec les diverses méthodes de stimulation de la signalisation Notch, la meilleure méthode dépendra de la question d&#39;expérimentation. Ici, notre méthode préférée de la culture de fractions adhérentes et non-adhérentes de cellules de la moelle rincés à partir des os longs de souris sera présenté. Cette méthode présente l&#39;avantage de ne nécessiter pratiquement pas d&#39;équipement spécialisé et pour produire des cellules qui sont applicables à une variété de procédés de différenciation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Recombinant mouse M-CSF</td>
			<td>Biolegend</td>
			<td>576402</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Recombinant mouse RANKL</td>
			<td>Shenandoah Biotechnology</td>
			<td>200-04</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Recombinant human Jagged1-Fc</td>
			<td>R&#38;D Systems</td>
			<td>1277-JG-050</td>
			<td>Available from multiple suppliers, test activity before experiments</td>
		</tr>
		<tr>
			<td>Protein G agarose beads</td>
			<td>InvivoGen</td>
			<td>gel-agg-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-human IgG Fc</td>
			<td>Jackson ImmunoResearch</td>
			<td>109-001-008</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium powder</td>
			<td>Sigma-Aldrich</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase cell dissociation reagent</td>
			<td>ThermoFisher</td>
			<td>A1110501</td>
			<td>Used to lift osteoclast precursors</td>
		</tr>
		<tr>
			<td>Acid Phosphatase, Leukocyte (TRAP) Kit</td>
			<td>Sigma-Aldrich</td>
			<td>387A-1KT</td>
			<td>Used to stain differentiated osteoclasts</td>
		</tr>
	</tbody>
</table>

stimulation of notch signaling in mouse osteoclast precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Le but de cette méthode est de la culture et de stimuler la signalisation Notch dans les précurseurs des ostéoclastes. Lorsqu&#39;il est correctement cultivés, les précurseurs d&#39;ostéoclastes présentent une morphologie fusiforme essentiellement allongée avec cytoplasme lisse (figure 1A). Des précautions doivent être prises pour éviter l&#39;activation immunologique des précurseurs d&#39;ostéoclastes. Lors de l&#39; activation, les précurseurs se propagen...

Watch this Scientific Journal Video about Stimulation of Notch Signaling in Mouse Osteoclast Precursors at JoVE.com

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Stimulation de Notch Signalisation dans Souris ostéoclastes Précurseurs

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

The overall goal of this procedure is to observe the affects of notch signaling stimulation at varying points during the osteoclast differentiation process. This method can help answer key questions in the field of cell biology, such as:How does the role of notch signaling change during a cellular process? The main advantage of this technique is its marginality, which allows it to be adapted to other cells or notch ligands.While this method can provide insight into osteoclast differentiation, it can also be applied to other processes such as cell survival, proliferation, or to the differentiation of other cell types. Begin the procedure by securing a mouse in the supine position and disinfecting the skin with 70 percent ethanol. Next, make an incision along the anterior surface of one hind limb to expose the tibia and femur.Followed by an incision through the knee joint to free the distal end of the femur. Remove the femur by cutting through the end of the bone close to the pelvis and clear away any of the attached soft tissue. Cut through the ankle to remove the tibia and remove the soft tissue.Then, use forceps to hold the leg bone over a 15 milliliter conical tube and insert a 25 and five-eighths gauged needle attached to a 10 milliliter syringe into the uncut end of the bone to flush two point five milliliters of room temperature alpha MEM through the cut end of the bone into the tube. When all of the bone marrow has been removed, the bone will turn a clear, beige color. After collecting the marrow from each bone, allow any large debris to settle to the bottom of the tube and transfer the debris-free, bone marrow cell containing supernatant into a new 15 milliliter tube.Pellet the cells by centrifugation. Then lyse the red blood cells in zero point five milliliters of ACK Lysis buffer at 37 degrees Celsius for three minutes. At the end of the incubation, restore the isotonicity with 10 milliliters of PBS and collect the cells by centrifugation.Resuspend the pellet in 10 milliliters of fresh Alpha MEM for plating. In the 100 milliliter tissue culture treated dish, promouse. And incubate the cells overnight at 37 degrees Celsius in a humidified tissue-culture incubator.After enriching for the osteoclast precursors, wash the cells in five milliliters of PBS and treat them for three to five minutes with one milliliter of cell-dissociation reagent at room temperature until the osteoclast precursors can be dislodged with gentle tapping. Next, add four milliliters of Alpha MEM to the dish and transfer the cells to a 15 milliliter conical tube. After counting, dilute the precursors to a five times 10 to the fourth cells per milliliter concentration in Alpha MEM.And treat them with MCSF and RANKL, then immediately seed one milliliter of cells into each well of a 24 well tissue-treated plate. Further incubation and 37 degrees Celsius in the humidified tissue culture incubator. After two days, replace the medium with one milliliter of fresh osteoclast medium to continue the differentiation.For continuous notch signaling simulation, coat single well of a 24 well plate with 250 microliters of goat anti-human, IGG FC antibody in PBS. After one hour at room temperature, remove any unbound antibody with three washes and one milliliter of PBS. Next, add jagged1-FC fusion protein in PBS to the well for a two hour incubation at room temperature.At the end of the incubation, wash the well three times with fresh PBS as just demonstrated. After the third wash, remove the PBS and immediately plate two point six times 10 to the fourth osteoclast precursors per centimeter squared onto the coated surface. Then transfer the plate to the tissue culture incubator for continuous notch signaling for up to three days.For temporary notch signaling stimulation, first add one microgram of jagged1-FC with 40 microliters of protein G Agarose beads in one point five milliliters of PBS for two hour incubation with rotation at four degrees Celsius. At the end of the incubation, collect the beads by centrifugation followed by two washes and one point five milliliters of PBS under the same centrifuge conditions. After the second wash, re-suspend the jagged1 conjugated beads in 130 microliters per centimeter squared of culture medium and add the entire volume of beads directly onto two point six times 10 to the fourth osteoclast precursors in one well of a 24 well plate for their cold culture at 37 degrees Celsius and five percent carbon dioxide.After the appropriate experimental stimulation period, remove the beads with several washes of PBS. Notch signaling activation can then be assayed by measuring the target gene transcription. When properly cultured, osteoclast precursors exhibit a primarily elongated, spindle-shaped morphology with a smooth cytoplasm.Upon activation, the precursors spread and become flattened with foamy cytoplasm obtaining a resistance to RANK signaling and do not efficiently differentiate into mature osteoclasts. Under the correct culture and differentiation conditions, enriched osteoclast precursors will differentiate and infuse into large, TRAP-positive, multi-nuclear osteoclasts within three days of treatment. Seeding osteoclast precursors onto jagged1 FC-coated surfaces for their continuous stimulation induces the upper regulation of the expression of specific notch-target genes within 24 hours, while the expression of other notch target genes is not significantly altered by the stimulation.Temporary notch signaling stimulation, via jagged1 FC coated beads with as little as 600 nanograms of jagged1 FC protein also induces a significant increase in the expression of the specific notch target genes. While attempting this procedure, it's important to remember that a common cause of osteoclast precursor differentiation failure is by accidental activation during handling. This can be assessed by measuring specific activation markers such as TNF to Inos.After watching this video, you should have a good idea of how to differentiate osteoclast precursors and also to stimulate notch signaling, both transiently and continuously.

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Live Imaging of Mouse Secondary Palate Fusion

Imagerie en direct de la souris Fusion de palais secondaire

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

Une nouvelle utilisation d’échographie en trois dimensions de haute fréquence pour la caractérisation de grossesse précoce chez la souris

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

Une base RANKL Osteoclast Culture dosage de moelle osseuse de souris pour enquêter sur le rôle de mTORC1 dans la Formation des ostéoclastes

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Isolement, Culture et différenciation des cellules stromales de la moelle osseuse et les ostéoclastes progéniteurs de souris

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cellule des épreuves d’agrégation pour évaluer la liaison de l’encoche de la drosophile avec Trans-Ligands et son Inhibition par Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Suppression de la signalisation profibrotiques potentialise médiée par les facteurs reprogrammation des fibroblastes embryonnaires de souris dans les Cardiomyocytes induites

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Visualiser le nœud et la notochorde plaque en Gastrulating des embryons de souris à l’aide de la microscopie électronique à balayage et toute monture Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...