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).

All research involving vertebrate animals was performed in accordance with protocols approved by the University of Pennsylvania Institutional Animal Care and Use Committee (IACUC).
1. Culture Media Preparation
<ol>
	<li>Prepare &#945;-MEM by dissolving minimum essential medium (MEM) powder and 1.9 g sodium bicarbonate in 900 ml H2O and supplement with 2 mM L-glutamine, 100 units/ml penicillin, 100 &#956;g/ml streptomycin, and 100 ml heat-inactivated fetal bovine serum. Sterilize u...

<ol>
	<li>Harper, J. A., Yuan, J. S., Tan, J. B., Visan, I., Guidos, C. J. <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+in+development+and+disease.">Notch signaling in development and disease.</a> Clin Genet. 64 (6), 461-472 (2003).</li><li>Andersson, E. R., Sandberg, R., Lendahl, U. <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:+simplicity+in+design,+versatility+in+function.">Notch signaling: simplicity in design, versatility in function.</a> Development. 138 (17), 3593-3612 (2011).</li><li>Meloty-Kapella, L., Shergill, B., Kuon, J., Botvinick, E., Weinmaster, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+ligand+endocytosis+generates+mechanical+pulling+force+dependent+on+dynamin,+epsins,+and+actin.">Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin.</a> Dev Cell. 22 (6), 1299-1312 (2012).</li><li>Chowdhury, F., 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=Defining+Single+Molecular+Forces+Required+for+Notch+Activation+Using+Nano+Yoyo.">Defining Single Molecular Forces Required for Notch Activation Using Nano Yoyo.</a> Nano Lett. 16 (6), 3892-3897 (2016).</li><li>Chillakuri, C. R., Sheppard, D., Lea, S. M., Handford, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+receptor-ligand+binding+and+activation:+insights+from+molecular+studies.">Notch receptor-ligand binding and activation: insights from molecular studies.</a> Semin Cell Dev Biol. 23 (4), 421-428 (2012).</li><li>Vas, V., Szilagyi, L., Paloczi, K., Uher, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+Jagged-1+is+able+to+inhibit+the+function+of+its+multivalent+form+to+induce+hematopoietic+stem+cell+self-renewal+in+a+surrogate+in+vitro+assay.">Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay.</a> J Leukoc Biol. 75 (4), 714-720 (2004).</li><li>Small, D., 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=Soluble+Jagged+1+represses+the+function+of+its+transmembrane+form+to+induce+the+formation+of+the+Src-dependent+chord-like+phenotype.">Soluble Jagged 1 represses the function of its transmembrane form to induce the formation of the Src-dependent chord-like phenotype.</a> J Biol Chem. 276 (34), 32022-32030 (2001).</li><li>Goncalves, R. M., Martins, M. C., Almeida-Porada, G., Barbosa, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+notch+signaling+by+immobilization+of+jagged-1+on+self-assembled+monolayers.">Induction of notch signaling by immobilization of jagged-1 on self-assembled monolayers.</a> Biomaterials. 30 (36), 6879-6887 (2009).</li><li>Varnum-Finney, B., 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+Notch+ligand,+Jagged-1,+influences+the+development+of+primitive+hematopoietic+precursor+cells.">The Notch ligand, Jagged-1, influences the development of primitive hematopoietic precursor cells.</a> Blood. 91 (11), 4084-4091 (1998).</li><li>Zhu, F., Sweetwyne, M. T., 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=PKCdelta+is+required+for+Jagged-1+induction+of+human+mesenchymal+stem+cell+osteogenic+differentiation.">PKCdelta is required for Jagged-1 induction of human mesenchymal stem cell osteogenic differentiation.</a> Stem Cells. 31 (6), 1181-1192 (2013).</li><li>Yang, B., 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=Effect+of+radiation+on+the+Notch+signaling+pathway+in+osteoblasts.">Effect of radiation on the Notch signaling pathway in osteoblasts.</a> Int J Mol Med. 31 (3), 698-706 (2013).</li><li>Urs, 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=Effect+of+soluble+Jagged1-mediated+inhibition+of+Notch+signaling+on+proliferation+and+differentiation+of+an+adipocyte+progenitor+cell+model.">Effect of soluble Jagged1-mediated inhibition of Notch signaling on proliferation and differentiation of an adipocyte progenitor cell model.</a> Adipocyte. 1 (1), 46-57 (2012).</li><li>LeComte, M. 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. K., Zanotti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hajdu+Cheney+Mouse+Mutants+Exhibit+Osteopenia,+Increased+Osteoclastogenesis,+and+Bone+Resorption.">Hajdu Cheney Mouse Mutants Exhibit Osteopenia, Increased Osteoclastogenesis, and Bone Resorption.</a> J Biol Chem. 291 (4), 1538-1551 (2016).</li></ol>

Critical steps within the protocol
Culture and in vitro differentiation of osteoclast precursors provides a useful platform for investigation of molecular mechanisms of osteoclastogenesis and identification of therapeutic targets for bone regeneration and preservation of bone mass. When culturing mouse osteoclast precursors, the most critical element is maintenance of precursors in a na&#239;ve state. As macrophage-like cells, osteoclast precursors are primed to respond to bacterial comp...

The mammalian Notch signaling pathway is homologous to the same pathway in Drosophila melanogaster and consists of four transmembrane Notch receptors (Notch1-4) and five membrane-bound ligands of the Jagged (JAG1 &#38; JAG2) and Delta-like (DLL1, 3,&#38; 4) families1. Notch signaling is initiated when Notch receptor on a receiving cell is bound by ligand on a transmitting cell2. During this trans-activation, the membrane-bound Notch ligand produces a stretching force on the membrane-bound Notch receptor3,4. The stretching force of ligand binding induces conformational changes in the Notch receptor that facilitate extracellular cleavage of the receptor by TNFalpha converting enzyme (TACE) followed by an intracellular cleavage event mediated by a presenilin-containing gamma-secretase complex (&#947;-secretase). &#947;-secretase releases the Notch intracellular domain (NICD) which translocates into the nucleus where it forms a transcriptional activation complex with CBF-1-Su(H)-Lag-1 (CSL), mastermind-like (MAML), and cell type-specific factors to drive expression of target genes5.
The mechanical elements of Notch signaling activation result in the need for unique methods of Notch pathway activation in vitro. Soluble Notch ligands can bind to Notch receptors, but fail to produce stretching forces necessary for NICD release while at the same time competitively inhibiting binding of cell-associated Notch ligands. Thus, addition of soluble Notch ligands to culture medium can attenuate normal Notch signaling6,7. Fortunately, Notch ligands can induce NICD release if they are fixed to a suitably rigid substrate5,8,9,10. Seeding cells on ligand-coated culture substrates or applying ligand-coated beads to cells can both activate Notch signaling, and the choice between them depends primarily on the desired timing of Notch stimulation. For immediate, temporary Notch signaling activation, as would be desired during the midpoint of a functional or differentiation assay, Notch ligand can be bound to agarose beads, applied to cultured cells, and washed out at any time. For more sustained Notch signaling from the beginning of a culture period, tissue culture plates can be coated with ligand prior to cell seeding.
For the purposes of this protocol, methods are carried out using mouse osteoclast precursors, but the methods and variations on the methods described here are applicable to a wide variety of cell types6,11,12,13,14. Osteoclasts are terminally differentiated hematopoietic linage cells that are responsible for the resorption of bone tissue, and they are implicated in multiple disorders of bone loss15. Thus, in vitro study of the differentiation of osteoclasts from their monocyte/macrophage-lineage precursors and the molecular mechanisms controlling their function is essential to better understanding of osteoclasts and development of new bone-regenerating therapies. While it is now generally accepted that Notch signaling plays a positive role in the differentiation and function of osteoclasts, variations in both Notch signaling stimulation and osteoclast precursor culture and differentiation led to initially contradictory findings16,17,18,19. Closer examination of the differences in methods and use of genetic models have greatly clarified the role of Notch signaling in osteoclastogenesis, but application of standardized Notch stimulation and culture methods could prevent such controversies in future studies of Notch signaling in other cell types20,21,22,23.
There are multiple methods for culturing and differentiating mouse osteoclast precursors, and, as with varying methods for stimulating Notch signaling, the best method will depend upon the experimental question. Herein, our preferred method of culturing adherent and non-adherent fractions of marrow cells flushed from mouse long bones will be presented. This method has the advantage of requiring essentially no specialized equipment and producing cells that are applicable to a variety of differentiation methods.

<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.

The aim of this method is to culture and stimulate Notch signaling in osteoclast precursors. When properly cultured, osteoclast precursors exhibit a primarily elongated spindle-shaped morphology with smooth cytoplasm (Figure 1A). Care should be taken to avoid immunological activation of the osteoclast precursors. Upon activation, precursors spread and become flattened with foamy cytoplasm (Figure 1B). These &#34;fried egg&#34; cells are resistant to RANK ...

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.

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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