CINXE.COM

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class="clear"></div> </div> <div class="container_12"> <div class="grid_9 base_rnd_css"> <div class="region region-content"> <div id="block-et-messages-et-messages-block" class="block block-et-messages"> <div class="content"> <span></span> </div> </div> <div id="block-system-main" class="block block-system"> <div class="content"> <div id="node-2821" class="node node-protocols clearfix" about="/resources/protocols/adipogenic-differentiation-humanmouse-mesenchymal-stem-cells" typeof="sioc:Item foaf:Document"> <span property="dc:title" content="Adipogenic Differentiation of Human/Mouse Mesenchymal Stem Cells" class="rdf-meta element-hidden"></span><span property="sioc:num_replies" content="0" datatype="xsd:integer" class="rdf-meta element-hidden"></span> <div class="content"> <div class="field field-name-body field-type-text-with-summary field-label-hidden"><div class="field-items"><div class="field-item even" property="content:encoded"><p>Mesenchymal stem cells (MSCs) are functionally defined by their capacity to self renew and their ability to differentiate into multiple cell types including adipocytes, chondrocytes, and osteocytes. This protocol describes the adipogenic differentiation of human and mouse MSCs using the <a href="/products/stemxvivo-osteogenic-adipogenic-base-media_ccm007">StemXVivo™ Human/Mouse Osteogenic/Adipogenic Base Media</a> (Catalog # CCM007) and <a href="/products/stemxvivo-adipogenic-supplement-100x-_ccm011">StemXVivo Adipogenic Supplement</a> (Catalog # CCM011).</p> <p>Please read the protocol in its entirety before starting.</p> <h3>Supplies Required</h3> <h4>Reagents</h4> <ul> <li>Bone marrow derived MSCs</li> <li><a href="/products/stemxvivo-osteogenic-adipogenic-base-media_ccm007">StemXVivo Human/Mouse Osteogenic/Adipogenic Base Media</a> (R&D Systems, Catalog # CCM007)</li> <li>S<a href="/products/stemxvivo-adipogenic-supplement-100x-_ccm011">temXVivo Adipogenic Supplement</a> (R&D Systems, Catalog # CCM011)</li> <li>Penicillin-Streptomycin (100x)</li> </ul> <h4>Materials</h4> <ul> <li>10 cm tissue culture plates</li> <li>15 mL centrifuge tubes</li> <li>Serological pipettes</li> <li>Pipettes and pipette tips</li> </ul> <h4>Equipment</h4> <ul> <li>37° C, 5% CO<sub>2</sub> humidified incubator</li> <li>Centrifuge</li> <li>Hemocytometer</li> <li>Microscope</li> <li>Water bath</li> </ul> <h3>Reagent & Media Preparation</h3> <p>Note: Sterile technique is required when handling the reagents.</p> <ul> <li><strong>StemXVivo Osteogenic/Adipogenic Base Media</strong> - Thaw the StemXVivo Osteogenic/Adipogenic Base Media at 2 - 8° C or room temperature. Aliquot any unused thawed media and store at -20° C in a manual defrost freezer. Thawed media may be stored in the dark at 2 - 8° C for up to 1 month.</li> <li><strong>Completed StemXVivo Osteogenic/Adipogenic Base Media</strong> - Add Penicillin-Streptomycin to the StemXVivo Osteogenic/Adipogenic Base Media at a 1:100 dilution.<br /> Note: If Penicillin-Streptomycin is not needed for the experiment, it can be omitted.</li> <li><strong>Completed StemXVivo Adipogenic Differentiation Media</strong> - If a precipitate forms, warm the Adipogenic Supplement vial in a 37° C water bath for 5 minutes. Vortex until the precipitate dissolves. Add StemXVivo Adipogenic Supplement to the completed StemXVivo Osteogenic/Adipogenic Base Media at a 1:100 dilution.</li> </ul> <h3>Procedure</h3> <div class="Image" style="FLOAT: right; WIDTH: 269px"> <p><strong>Figure 1</strong></p> <img align="right" alt="Figure 1" height="498" src="//resources.rndsystems.com/images/site/figure1Adipogenic6178.gif" width="269" /></div> <p>Note: When handling biohazard materials such as human cells, safe laboratory procedures should be followed and protective clothing should be worn.</p> <ol> <li>Pre-warm the Completed StemXVivo Osteogenic/Adipogenic Base Media in a 37° C water bath. This procedure uses 10 mL for each 10 cm tissue culture plate used.</li> <li>Resuspend 1 x 10<sup>6 </sup>MSCs into 10 mL of the pre-warmed Completed StemXVivo Osteogenic Base Media.<br /> Note: If using another size tissue culture vessel, seed cells at approximately 2.1 x 10<sup>4</sup> cells/cm<sup>2</sup>/0.2 - 0.3 mL.</li> <li>Add this cell suspension to a 10 cm tissue culture plate and incubate overnight in a 37° C, 5% CO<sub>2</sub> incubator. Cells should be 100% confluent after overnight incubation. If they are not confluent, replace media every 2 - 3 days with Osteogenic/Adipogenic Base Media until 100% confluency is reached.</li> <li>At 100% confluency, replace the media with 10 mL of pre-warmed completed StemXVivo Adipogenic Differentiation Media to induce adipogenesis.</li> <li>Every 3 - 4 days remove and discard the spent media and replace with 10 mL of freshly prepared, pre-warmed Completed StemXVivo Adipogenic Differentiation Media.<br /> Note: Differentiation is complete after 7 - 21 days, at which time adipogenic induced cells will have morphological changes and lipid vacuoles (Figure 2) and express markers of the adipocyte lineage (Figure 3). <table cellpadding="5" cellspacing="0" class="border" width="500"> <tbody> <tr> <td><img alt="Figure 2" src="//resources.rndsystems.com/images/site/adipogenic_ccm0076179.jpg" width="250" /></td> <td><img alt="Figure 3" src="//resources.rndsystems.com/images/site/adipogenic_sc0106180.jpg" width="250" /></td> </tr> <tr> <td valign="top"><strong>Figure 2. Adipogenic Differentiation of Human MSCs.</strong> Human mesenchymal stem cells differentiated <em>in vitro</em> for 21 days using the <a href="/products/stemxvivo-osteogenic-adipogenic-base-media_ccm007">StemXVivo Human/Mouse Osteogenic/Adipogenic Base Media</a> (Catalog # CCM007) and<a href="/products/stemxvivo-adipogenic-supplement-100x-_ccm011"> StemXVivo Human/Mouse Adipogenic Supplement</a> (Catalog # CCM011) were stained for the adipogenic lineage-specific marker, FABP-4 using a <a href="/products/human-fabp4-a-fabp-antibody_af3150">Goat Anti-Human FABP-4 Antigen Affinity-purified Polyclonal Antibody</a> (Catalog # AF3150) and a <a href="/products/donkey-anti-goat-igg-northernlights-nl557-conjugated-antibody_nl001">NorthernLights 557-conjugated Anti-Goat Secondary Antibody</a> (Catalog # NL001; red) and counterstained with DAPI (blue).</td> <td valign="top"><strong>Figure 3. Adipogenic Differentiation of Mouse MSCs.</strong> The <a href="/products/mouse-mesenchymal-stem-cell-functional-identification-kit_sc010">Mouse Mesenchymal Stem Cell Functional Identification Kit</a> (Catalog # SC010) was used to differentiate mouse MSCs into the adipogenic lineage. Mature differentiated cells were labeled using the adipogenic lineage-specific antibody to FABP4 supplied with the kit (red). The nuclei were counterstained with DAPI (blue). Similar results may be obtained using the <a href="/products/stemxvivo-osteogenic-adipogenic-base-media_ccm007">StemXVivo Human/Mouse Osteogenic/Adipogenic Base Media</a> (Catalog # CCM007) and<a href="/products/stemxvivo-adipogenic-supplement-100x-_ccm011"> StemXVivo Human/Mouse Adipogenic Supplement</a> (Catalog # CCM011).</td> </tr> </tbody> </table> </li> </ol> </div></div></div><div class="field field-name-field-category field-type-taxonomy-term-reference field-label-hidden"><div class="field-items"><div class="field-item even"><a href="/protocol-types/stem-cell-protocols" typeof="skos:Concept" property="rdfs:label skos:prefLabel">Stem Cell Protocols</a></div></div></div> </div> </div> </div> </div> <div id="block-et-relatedcontent-menu-et-relatedcontent-menu-block" class="block block-et-relatedcontent-menu"> <div class="content"> <div class="relatedcontent_links"><h3>Related Information</h3></div> <div class="row evenheight"><div class="col-md-6 col-xs-12"><a href="https://www.rndsystems.com/products/mesenchymal-stem-cells" class="btn btn-senary btn-relatedinfo" itemprop="relatedLink">Mesenchymal Stem Cells</a></div><div class="col-md-6 col-xs-12"><a href="https://www.rndsystems.com/products/stem-cell-products" class="btn btn-senary btn-relatedinfo" itemprop="relatedLink">Stem Cell Products</a></div></div> </div> </div> <div id="block-block-26" class="block block-block"> <div class="content">  <div class="embeddedServiceInvitation" id="snapins_invite" aria-live="assertive" role="dialog" aria-atomic="true" style="display: none;"> <div class="embeddedServiceInvitationHeader" aria-labelledby="snapins_titletext" aria-describedby="snapins_bodytext" style="height: 48px;"> <img id="embeddedServiceAvatar" /> <span class="embeddedServiceTitleText" id="snapins_titletext">Need help?</span> <button type="button" id="closeInvite" class="embeddedServiceCloseIcon" aria-label="Exit invitation">×</button> </div> <div class="embeddedServiceInvitationBody"> <p id="snapins_bodytext">How can we help you?</p> </div> <div class="embeddedServiceInvitationFooter" aria-describedby="snapins_bodytext"> <button type="button" class="embeddedServiceActionButton" id="rejectInvite">Close</button> <button type="button" class="embeddedServiceActionButton" id="acceptInvite">Start Chat</button> </div> </div> </div> </div> </div> </div> <div class="grid_3"> <div class="column_menu"> <a href="/resources/literature" class="parent">Literature</a><div class="right_menu_contents hide"><ul class="menu"><li class="first leaf"><a href="/resources/articles" title="" id="menu-literature-29336">Articles</a></li> <li class="leaf"><a href="/resources/literature" title="" id="menu-literature-29341">Literature</a></li> <li class="last leaf"><a href="/resources/posters" title="" id="menu-literature-29346">Posters</a></li> </ul></div><a href="/Products" class="parent">Product Categories</a><div class="right_menu_contents hide"><ul class="menu"><li class="first collapsed"><a href="/products/proteins" id="menu-products-12696">Recombinant Proteins</a></li> <li class="leaf"><a href="/products/good-manufacturing-practices-gmp-grade-proteins" id="menu-products-112931">GMP Proteins</a></li> <li class="leaf"><a href="/products/enzymes" id="menu-products-121111">Enzymes</a></li> <li class="leaf"><a href="/products/small-molecules-peptides" id="menu-products-112711">Small Molecules & Peptides</a></li> <li class="leaf"><a href="/products/immunotherapy" id="menu-products-121081">Immunotherapy</a></li> <li class="leaf"><a href="/products/elispot-kits-fluorospot-kits" id="menu-products-12681">ELISpots</a></li> <li class="leaf"><a href="/products/cdna-clones" title="R&D Systems offers large selections of cDNA Clones and expression-ready vectors. 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