We thank Haruya Ohno, Kosaku Shinoda, Louis Sharp, Emi Tomoda, and Lauren Ruiz for discussion, technical help and editorial assistance on the manuscript. This work was supported by grants from the NIH (DK087853), from the Program for Breakthrough Biomedical Research and from Asubio Pharm Inc. to S.K. U.L.A. was supported by a SHARE PhD fellowships from The University of Copenhagen and the EU FP7 project DIABAT (HEALTH-F2-2011-278373) to Lise Madsen and Karsten Kristiansen. We also acknowledge the DERC center grant (NIH P30 DK063720).

1. Prepare Digestion Medium
Make 5 ml per 5 mice per tissue (approximately 1 ml/1 g adipose tissue).
<ol>
 <li>Weigh in digestion enzymes:
 - Collaginase D: 1.5 u/ml (1108874103, 1 g, Roche, 70334223)
 - Dispase II: 2.4 u/ml (04942078001, 0,980 mg/lyo, Roche, 11466200)
 </li>
 <li>Add 25 ml PBS and mix well to dissolve</li>
 <li>Add CaCl2 just prior to digestion of the tissue at a final concentration of 10 mM</li>
</ol>
2. Dissect Adipose Tissue from Mice
<ol>
 <li>Dissection should be performed carefully, and immediately after the mice (6-8 weeks old) are sacrificed. Work sterile with ethanol sprayed on the mouse fur before opening it. Place the tissue directly in clean PBS, separate interscapular BAT and subcutaneous inguinal WAT.</li>
 <li>For interscapular BAT: have the mouse placed with the back faced up, cut open along the back and all the way to the neck. The brown adipose tissue can be found right under the skin between the shoulders (interscapular). BAT can be seen as two lobes, butterfly shaped. A thin layer of white fat covering the BAT should to be carefully removed.</li>
 <li>For subcutaneous WAT (inguinal WAT): remove skin. Inguinal WAT is found directly under the skin on both sides, starting almost on the back and going alongside, inside of the thighs and down toward the testicle.</li>
</ol>
3. Cut and Digest Adipose Tissue
<ol>
 <li>Quickly remove all contamination, like hair, skeletal muscle and connective tissue from the tissue sections.</li>
 <li>Dry tissue quickly on paper to not dilute dissection medium with PBS and place it on a dry plate.</li>
 <li>Add digestion medium (added CaCl2 prior to digestion) and mince the tissue into small pieces. Mix very well by pipetting up and down with a 5 ml pipette. Transfer the minced tissues in 50 ml tubes with the rest of the digestion medium, mix even more by pipetting up and down.</li>
 <li>Digest in 37 &deg;C with constant agitation at 150 rpm for 40-50 min. Check every 10-15 min to make sure the digestion is going well and to prevent over-digestion.</li>
</ol>
Note: Correct digestion medium and time are important. The tissue needs to be well digested, but excess digestion can damage the cells.
4. Filter Cell Suspension
<ol>
 <li>Stop digestion by adding 5 ml complete medium (DMEM/F12 containing 10% FPS and P/S). The cells should almost be completely homogenous. Mix well by pipetting.</li>
 <li>Centrifuge at 700 x g for 10 min.</li>
 <li>SVF can now be seen as a brownish pellet on the bottom of the tube. Aspirate the oily mature adipocyte layer on top and most of the liquid layer - keep the pellet and dissolve it in 10 ml complete medium. Mix well.</li>
 <li>Place a cell strainer (50-70 &mu;m diameter) over a new 50 ml tube and filter the cell suspension.</li>
</ol>
5. Plate Cells 
<ol>
 <li>Transfer cell suspension to a 15 ml tube and centrifuge at 700 x g for 10 min.</li>
 <li>Aspirate medium and re-suspend pellet in 10 ml complete medium. Pipette and mix well.</li>
 <li>Estimate how many plates are needed. This may vary and depends on the pellet size. A general rule is 2 of 10 cm plates from 5 mice for inguinal WAT and one 10 cm plate for BAT.</li>
 <li>Plate cells on collagen coated dishes (R&amp;D) in complete medium</li>
 <li>1-2 hr after plating the cells, aspirate medium, wash with PBS twice and add fresh medium. This step is important as this can remove red blood cells, immune cells and other contaminants.</li>
</ol>
6. Differentiate Cells
<ol>
 <li>Make maintenance and induction mediums.</li>
</ol>
Maintenance medium
Complete medium with added: 
Insulin, final conc. 5 &mu;g/ml (5 mg/ml stock, ***100 &mu;l of acetic acid in 10 ml H2O to prepare acidified water pH 2.5. dissolve insulin in the acidified water. Store stock at -20 &deg;C)
3,3',5-Triiodo-L-thyronine (T3), final conc. 1 nM (10 &mu;M stock, ***dissolve T3 in 1N NaOH and add medium to make 10 &mu;M stock. Sigma cat# T-2877)
Store at 4 &deg;C, good for one week
Induction medium
Maintenance medium containing following compounds:
Indomethacin, final conc. 125 &mu;M (0.125 M stock in ethanol, Sigma cat#I-7378). Indomethacin must be heated to 60 &deg;C to be dissolved.
Dexamethasone, final conc. 2 &mu;g/ml (2 mg/ml sock in ethanol, Sigma cat# D-1756) 3-Isobutyl-1-methylxanthine (IBMX), final conc. 0.5 mM (0.25 M stock in DMSO, Sigma cat# I-5879) Rosiglitazone, final conc. 0.5 &mu;M (10 mM stock in DMSO, Sigma cat# R-2408)
Note: Make fresh induction medium each time.
<ol start="2">
 <li>Grow primary adipocytes to 95-97% confluence in complete medium (confluent but not too packed)</li>
 <li>Change regular complete medium with induction medium (day 0)</li>
 <li>After 48 hr (day 2), change medium to maintenance medium with rosiglitazone (0.5 &mu;M)</li>
 <li>After additional 48 hr (day 4), change to fresh maintenance medium with rosiglitazone (1 &mu;M) for additional 2-3 days. 6-7 days after adding the induction medium, cells are fully differentiated to mature fat cells and are filled with oil droplets. Droplets will appear around 3-4 days after the induction.</li>
</ol>
Note: Change medium every 2-3 days until the cells are fully differentiated.
Cells can now be harvested and mRNA expression of Ucp1 and other brown adipocyte-specific genes can be measured by qRT-PCR. Western blot will be used to detect proteins.

<ol>
	<li>Barness, L. A., Opitz, J. M., Gilbert-Barness, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity:+genetic,+molecular,+and+environmental+aspects.">Obesity: genetic, molecular, and environmental aspects.</a> American Journal of Medical Genetics. Part A. 143A, 3016-3034 (2007).</li><li>Rothwell, N. J., Stock, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+effects+of+cafeteria+and+tube-feeding+on+energy+balance+in+the+rat.">Combined effects of cafeteria and tube-feeding on energy balance in the rat.</a> The Proceedings of the Nutrition Society. 38, 5A (1979).</li><li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brown+adipose+tissue:+function+and+physiological+significance.">Brown adipose tissue: function and physiological significance.</a> Physiological Reviews. 84, 277-359 (2004).</li><li>Cypess, A. 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=Identification+and+importance+of+brown+adipose+tissue+in+adult+humans.">Identification and importance of brown adipose tissue in adult humans.</a> The New England Journal of Medicine. 360, 1509-1517 (2009).</li><li>van Marken Lichtenbelt, W. 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=Cold-activated+brown+adipose+tissue+in+healthy+men.">Cold-activated brown adipose tissue in healthy men.</a> The New England Journal of Medicine. 360, 1500-1508 (2009).</li><li>Virtanen, K. 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=Functional+brown+adipose+tissue+in+healthy+adults.">Functional brown adipose tissue in healthy adults.</a> The New England Journal of Medicine. 360, 1518-1525 (2009).</li><li>Zingaretti, M. 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=The+presence+of+UCP1+demonstrates+that+metabolically+active+adipose+tissue+in+the+neck+of+adult+humans+truly+represents+brown+adipose+tissue.">The presence of UCP1 demonstrates that metabolically active adipose tissue in the neck of adult humans truly represents brown adipose tissue.</a> FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology. 23, 3113-3120 (2009).</li><li>Saito, 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=High+incidence+of+metabolically+active+brown+adipose+tissue+in+healthy+adult+humans:+effects+of+cold+exposure+and+adiposity.">High incidence of metabolically active brown adipose tissue in healthy adult humans: effects of cold exposure and adiposity.</a> Diabetes. 58, 1526-1531 (2009).</li><li>Seale, P., 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=PRDM16+controls+a+brown+fat/skeletal+muscle+switch.">PRDM16 controls a brown fat/skeletal muscle switch.</a> Nature. 454, 961-967 (2008).</li><li>Petrovic, N., 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=Chronic+peroxisome+proliferator-activated+receptor+gamma+(PPARgamma)+activation+of+epididymally+derived+white+adipocyte+cultures+reveals+a+population+of+thermogenically+competent,+UCP1-containing+adipocytes+molecularly+distinct+from+classic+brown+adipocytes.">Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes.</a> The Journal of Biological Chemistry. 285, 7153-7164 (2010).</li><li>Coulter, A. A., Bearden, C. M., Liu, X., Koza, R. A., Kozak, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+fat+interacts+with+QTLs+controlling+induction+of+Pgc-1+alpha+and+Ucp1+during+conversion+of+white+to+brown+fat.">Dietary fat interacts with QTLs controlling induction of Pgc-1 alpha and Ucp1 during conversion of white to brown fat.</a> Physiological Genomics. 14, 139-147 (2003).</li><li>Klingenspor, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cold-induced+recruitment+of+brown+adipose+tissue+thermogenesis.">Cold-induced recruitment of brown adipose tissue thermogenesis.</a> Experimental Physiology. 88, 141-148 (2003).</li><li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adipose+organ.">The adipose organ.</a> Prostaglandins, leukotrienes, and essential fatty acids. 73, 9-15 (1016).</li><li>Ghorbani, M., Himms-Hagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appearance+of+brown+adipocytes+in+white+adipose+tissue+during+CL+316,243-induced+reversal+of+obesity+and+diabetes+in+Zucker+fa/fa+rats.">Appearance of brown adipocytes in white adipose tissue during CL 316,243-induced reversal of obesity and diabetes in Zucker fa/fa rats.</a> International Journal of Obesity and Related Metabolic Disorders: Journal of the International Association for the Study of Obesity. 21, 465-475 (1997).</li><li>Hansen, J. B., Kristiansen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+circuits+controlling+white+versus+brown+adipocyte+differentiation.">Regulatory circuits controlling white versus brown adipocyte differentiation.</a> The Biochemical Journal. 398, 153-168 (2006).</li><li>Ohno, H., Shinoda, K., Spiegelman, B. M., Kajimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PPARy+agonists+induce+a+white-to-brown+fat+conversion+through+stabilization+of+PRDM16+protein.">PPARy agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein.</a> Cell metabolism. 15, 395-404 (2012).</li><li>Lee, Y. H., Petkova, A. P., Mottillo, E. P., Granneman, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+identification+of+bipotential+adipocyte+progenitors+recruited+by+beta3-adrenoceptor+activation+and+high-fat+feeding.">In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding.</a> Cell Metabolism. 15, 480-491 (2012).</li><li>Cinti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transdifferentiation+properties+of+adipocytes+in+the+adipose+organ.">Transdifferentiation properties of adipocytes in the adipose organ.</a> American Journal of Physiology. Endocrinology and Metabolism. 297, E977-E986 (2009).</li><li>Barbatelli, G., 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+emergence+of+cold-induced+brown+adipocytes+in+mouse+white+fat+depots+is+determined+predominantly+by+white+to+brown+adipocyte+transdifferentiation.">The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.</a> American Journal of Physiology. Endocrinology and Metabolism. 298, E1244-E1253 (2010).</li><li>Petrovic, N., 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=Chronic+peroxisome+proliferator-activated+receptor+gamma+(PPARgamma)+activation+of+epididymally+derived+white+adipocyte+cultures+reveals+a+population+of+thermogenically+competent,+UCP1-containing+adipocytes+molecularly+distinct+from+classic+brown+adipocytes.">Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes.</a> The Journal of Biological Chemistry. 285, 7153-7164 (2009).</li><li>Rong, J. X., 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=Adipose+mitochondrial+biogenesis+is+suppressed+in+db/db+and+high-fat+diet-fed+mice+and+improved+by+rosiglitazone.">Adipose mitochondrial biogenesis is suppressed in db/db and high-fat diet-fed mice and improved by rosiglitazone.</a> Diabetes. 56, 1751-1760 (2007).</li><li>Wilson-Fritch, L., 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=Mitochondrial+remodeling+in+adipose+tissue+associated+with+obesity+and+treatment+with+rosiglitazone.">Mitochondrial remodeling in adipose tissue associated with obesity and treatment with rosiglitazone.</a> The Journal of Clinical Investigation. 114, 1281-1289 (2004).</li><li>Sell, 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=Peroxisome+proliferator-activated+receptor+gamma+agonism+increases+the+capacity+for+sympathetically+mediated+thermogenesis+in+lean+and+ob/ob+mice.">Peroxisome proliferator-activated receptor gamma agonism increases the capacity for sympathetically mediated thermogenesis in lean and ob/ob mice.</a> Endocrinology. 145, 3925-3934 (2004).</li><li>Fukui, Y., Masui, S., Osada, S., Umesono, K., Motojima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+thiazolidinedione,+NC-2100,+which+is+a+weak+PPAR-gamma+activator,+exhibits+potent+antidiabetic+effects+and+induces+uncoupling+protein+1+in+white+adipose+tissue+of+KKAy+obese+mice.">A new thiazolidinedione, NC-2100, which is a weak PPAR-gamma activator, exhibits potent antidiabetic effects and induces uncoupling protein 1 in white adipose tissue of KKAy obese mice.</a> Diabetes. 49, 759-767 (2000).</li><li>Vernochet, 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=C/EBPalpha+and+the+corepressors+CtBP1+and+CtBP2+regulate+repression+of+select+visceral+white+adipose+genes+during+induction+of+the+brown+phenotype+in+white+adipocytes+by+peroxisome+proliferator-activated+receptor+gamma+agonists.">C/EBPalpha and the corepressors CtBP1 and CtBP2 regulate repression of select visceral white adipose genes during induction of the brown phenotype in white adipocytes by peroxisome proliferator-activated receptor gamma agonists.</a> Molecular and Cellular Biology. 29, 4714-4728 (2009).</li><li>Tai, T. 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=Activation+of+the+nuclear+receptor+peroxisome+proliferator-activated+receptor+gamma+promotes+brown+adipocyte+differentiation.">Activation of the nuclear receptor peroxisome proliferator-activated receptor gamma promotes brown adipocyte differentiation.</a> The Journal of Biological Chemistry. 271, 29909-29914 (1996).</li><li>Sugii, 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=PPARgamma+activation+in+adipocytes+is+sufficient+for+systemic+insulin+sensitization.">PPARgamma activation in adipocytes is sufficient for systemic insulin sensitization.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 22504-22509 (2009).</li></ol>

The authors declare that they have no competing financial interests.

Here we present a reliable cellular system to study the development of beige/brite cells in primary cultured adipocytes in mice. As compared to several available immortalized cell lines, this system is likely to offer enhanced relevance to the browning of white fat in vivo.
Even though the study of these primary adipocytes offers some advantages, there also exist some limitations and concerns that are important to consider. First, this system is highly reliant on differentiation poten...

Obesity is dramatically increasing worldwide and is now considered one of the most serious concerns to public health 1. This condition is related to a misbalance in energy intake relative to expenditure and results in excess energy stored as lipid in white adipose tissue (WAT). Enlarged WAT is associated with increased body mass and weight, while brown adipose tissue has the ability to dissipate excess energy to produce heat. Hence BAT can function as protection against both cold and obesity 2,3 . This is achieved by uncoupling of the electron transport in mitochondria by uncoupling protein 1 (UCP1). This protein is considered a hallmark for nonshivering thermogenesis in BAT3. Several studies in recent years revealed that adult humans have functional BAT 4-8 and, consequently, manipulation of BAT in humans can be a potential therapeutic intervention in the battle against obesity and its related diseases.
Current evidence indicates that two types of brown adipocytes exist in rodents; "classical" or "pre-existing" brown fat develops during the prenatal stage and forms dedicated brown adipose depots in interscapular region and other peripheral tissues. On the other hand, an "inducible" form of brown fat (so called brite or beige cells) develops during post-natal stage and appears interspersed in white adipose tissues. The two types of brown adipocytes are also separated by different developmental origins. While the pre-existing brown adipocytes arise from myoblastsic-like Myf5 precursors, the inducible brite/beige cells interspersed in WAT arise from a non-Myf5 lineage9,10 . In addition, regulatory pathways of this cell type is likely to be different from the Myf5-derived brown adipocytes 11. The development of the beige cells (i.e. "browning" of white fat) can be activated in response to chronic cold exposure and to &beta;3-adrenoceptor-agonists or PPAR&gamma; agonists in adults12-14. The beige/brite cells are likely to be a promising therapeutic target for manipulation of overall energy balance and can potentially become part of obesity treatment; hence, it is important to understand precise molecular mechanisms and signaling pathways by which environmental cues control the development of beige cells.
To understand the molecular control of the browning of white fat, in vitro experiments are best suited as differentiation of preadipocytes takes place rather asynchronously and it is difficult to detect the cells in situ 15. Although studies on adipocyte development have thus far been performed primarily on cell lines such as 3T3-L1, 3T3-F442A or HIB1, these cell lines appear to lack the molecular signature of beige cells. On the other hand, primary adipocytes isolated from subcutaneous WAT are most likely to recapitulate the process of browning of white fat in a cell autonomous fashion. Here we provide a protocol for effective isolation of the stromal-vascular fraction from adipose tissues and for inducing the browning of white fat in response to PPAR&gamma; agonists. Rosiglitazone has been shown to be an especially effective mediator of browning in these cells. As previously suggested 16, this cellular system can be used to serve a reliable cellular system to study the development of beige/brite cells.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Collaginase D</td>
 <td>Roche</td>
 <td>11088874103 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dispase II</td>
 <td>Roche</td>
 <td>04942078001 </td>
 <td></td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM medium</td>
 <td>Fisher</td>
 <td>10017-CV </td>
 <td>With 2,5 g/l glucose &amp; L-glutamine without sodium pyruvate </td>
 </tr>
 <tr>
 <td>Insulin</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>T3 (3,3',5-Triiodo-L-thyronine) </td>
 <td>Sigma </td>
 <td>T-2877 </td>
 <td></td>
 </tr>
 <tr>
 <td>Indomethacin </td>
 <td>Sigma </td>
 <td>I-7378 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dexamethasone </td>
 <td>Sigma </td>
 <td>D-1756</td>
 <td></td>
 </tr>
 <tr>
 <td>IBMX </td>
 <td>Sigma</td>
 <td>I-5879</td>
 <td></td>
 </tr>
 <tr>
 <td>Rosiglitazone </td>
 <td>Sigma</td>
 <td>R-2408</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Collagen coated dishes</td>
 <td>BD </td>
 <td>354450</td>
 <td>10 cm plates</td>
 </tr>
 <tr>
 <td> 70 &mu;m filter </td>
 <td>BD Falcon</td>
 <td>352350</td>
 <td>Cell strainer,70 &mu;m nylon 1/ea</td>
 </tr>
 </tbody>
</table>

isolation and differentiation of stromal vascular cells to beige/brite cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Browning of primary adipocytes can be accessed by measuring mRNA expression of Ucp1 and other brown fat-specific or selective genes by qRT-PCR. Presented in Figure 1 is gene expression data in inguinal WAT-derived primary adipocytes. The cells were induced to differentiate in the presence of two different doses of rosiglitazone at 50 nM and 500 nM, respectively. A subset of cells was treated with forskolin at 10 &mu;M for 4 hr prior to harvest. This will induce cyclic-AMP (cAMP) in the cells and...

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Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

isolation-differentiation-stromal-vascular-cells-to-beigebrite

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Biology

38.5K Aufrufe.  University of California, San Francisco. Primäre weißen Präadipozyten aus weißem Fettgewebe von Mäusen isoliert werden in beige / brite Zellen unterschieden werden. Präsentiert hier ist ein zuverlässiger zellulären Modellsystem, um die molekulare Regulation von "Bräunung" der weiße Fett zu studieren.

Serie: Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

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Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells