We thank Daniel Mondrag&#243;n, Antonio Prado, Fernando L&#243;pez-Barrera, Mart&#237;n Garc&#237;a-Serv&#237;n, Alejandra Castilla, and Mar&#237;a Antonieta Carbajo for their technical assistance, and Jessica Gonzalez Norris for critically editing the manuscript. This protocol was supported by Consejo Nacional de Ciencia y Tecnolog&#237;a de M&#233;xico (CONACYT), Fondo Sectorial de Investigacio&#769;n para la Educacio&#769;n&#160;Grant 284771 (to Y.M.).

All rodent experiments were approved by the Bioethics Committee of the Institute of Neurobiology of the UNAM, protocol number 075.
1. Isolation of mouse adipocyte precursor cells
<ol>
	<li>Euthanize 8-10-week-old male C57BL/6 mice (e.g., by CO2&#160;inhalation and subsequent cervical dislocation) (four animals per isolation). Disinfect the mice by rubbing with 70% ethanol and obtain the adipose tissue immediately after sacrifice. 
	NOTE: After the euthanasia of rodents, isolate the adipose tissue immediately and perform all steps inside a sterile laminar flow hood. Mice are kept under 12 h light/dark cycles with free access to food and water.</li>
	<li>Dissect the inguinal subcutaneous adipose tissue from each mouse. Remove only the adipose tissue, avoiding the removal of muscle, skin, or any other tissue, and collect it in a 50 mL conical tube containing 15 mL of type 1 collagenase solution (1.5 mg/mL) on ice. Dissolve type 1 collagenase in Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose-1% bovine serum albumin (BSA) and filter through 0.2 &#181;m syringe filters.</li>
	<li>Cut the adipose tissue into small pieces with sterile surgical scissors and digest the samples by incubation with type 1 collagenase solution at 37 &#176;C in an orbital shaker (150 rpm) for 30 min (place the tube containing fat and collagenase in a horizontal position for a greater shaking surface). Check the digestion every 10 min to make sure it works (tissue degradation is visible) and to prevent overdigestion (see Supplementary Figure S1).</li>
	<li>Filter using a 200 &#181;m mesh-syringe (previously autoclaved) to eliminate tissue not digested with collagenase. Pass the filter over the edge of the tube to drain as many cells as possible into the solution. Add 15 mL of cold DMEM-1% BSA to stop digestion and centrifuge at 400 &#215; g for 10 min at 4 &#176;C.</li>
	<li>Aspirate the top layer containing mature adipocytes and most of the liquid layer; avoid touching or disturbing the pellet. Add 20 mL of cold phosphate buffered saline (PBS)-2% fetal bovine serum (FBS) and resuspend the pellet. Centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant, aspirating first the upper layer to eliminate the remaining adipocytes and fat. Resuspend the pellet in 1 mL of ammonium chloride potassium (ACK) lysing buffer (to lyse red blood cells) and incubate on ice for 5 min. Add 10 mL of PBS-2% FBS, mix, and centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Aspirate the supernatant and resuspend the pellet in 200 &#181;L of anti-Fc solution (purified rat anti-mouse CD16/CD32 diluted in PBS-2% FBS [1:150]) to reduce Fc receptor-mediated binding by antibodies of interest. Incubate on ice for 5 min. Transfer the cell suspension to a 5 mL tube that fits into the prechilled racks of a magnetic cell separator and centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Eliminate the supernatant, add 200 &#181;L of the mixture of CD31(PECAM-1) monoclonal antibody (390)-biotin (1:100) and CD45 monoclonal antibody (30-F11)-biotin (1:100), mix well, and incubate on ice for 15 min (prepare antibody dilutions in anti-Fc solution; see Table 1). Add 400 &#181;L of PBS-2% FBS and centrifuge at 400 &#215; g for 5 min at 4 &#176;C. 
	NOTE: CD31 and CD45 are markers of endothelial cells and hematopoietic cells, respectively.</li>
	<li>Aspirate the supernatant and incubate in 100 &#181;L of anti-biotin microbeads (1:5) for 15 min on ice (prepare antibody dilutions in anti-Fc solution, see Table 1). Add 400 &#181;L of PBS-2% FBS and centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in 350 &#181;L of PBS-2% FBS. Remove cell aggregates or large particles from the single-cell suspensions with a preseparation filter (70 &#181;m). First, activate the filter with 100 &#181;L of PBS-2% FBS, pass the cell suspension through the filter (collect in a clean tube), and finally wash the filter with 100 &#181;L of PBS-2% FBS.</li>
	<li>Perform magnetic separation of cells using the negative separation strategy with a magnetic cell separator.
	<ol>
		<li>Place the sample in position A of the chill rack (ensure that the rack is precooled) and two empty tubes in position B and C to recover the non-labeled and labeled cells, respectively.</li>
		<li>Load the washing buffer and running buffer into the corresponding bottles before turning on the equipment. Program the equipment to perform the magnetic separation of cells with the DEPLETE protocol. 
		NOTE: This protocol is for depletion in standard mode for the removal of cells with normal antigen expression (in this case, cells labeled with anti-CD31 and anti-CD45), if recovery is the highest priority.</li>
		<li>In the separation section, select the number of samples to be separated and select the DEPLETE protocol. Press Run and start the separation. At the end of the program, recover the unlabeled cells and centrifuge at 400 &#215; g for 5 min at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Remove the supernatant and resuspend the pellet in 500 &#181;L of growth medium (Table 1).</li>
	<li>Seed the APCs in one well of a 12-well plate previously coated with 2.5% basement membrane matrix (approximately 50,000-75,000 cells are obtained). Add 400 &#181;L of 2.5% basement membrane matrix to cover the entire surface of each well. Immediately after removing the excess solution and leaving only a small layer, let the plate dry (without a lid) inside the laminar flow hood for at least 1 h. Incubate at 37 &#176;C in a 5% CO2 atmosphere. Change the medium every 48 h until the cells reach 80% confluence.</li>
	<li>At 80% confluence, remove the medium completely and wash with 350 &#181;L of PBS-2% FBS. Harvest the cells with 350 &#181;L of 0.05% trypsin-EDTA for 2 min at 37 &#176;C. Add 2 mL of growth medium and collect the cells in a new 50 mL conical tube, then centrifuge at 400 &#215; g for 5 min.</li>
	<li>Passage the APCs to 12-well plates (10,000-20,000 cells per well) previously coated with 2.5% basement membrane matrix. Incubate at 37 &#176;C with 5% CO2. Change the medium every 48 h until cells reach 80% confluence. 
	NOTE: APCs can be passaged once. Subsequently, they lose their ability to proliferate and differentiate. See the workflow for the APCs isolation process in Supplementary Figure S2.</li>
</ol>
2.&#160;Adipocyte differentiation and induction of insulin resistance
NOTE: Maintain cells at 37 &#176;C in 5% CO2&#160;and perform steps involving change of medium and treatments with TNF-&#945; and insulin inside a sterile hood.
<ol>
	<li>At 80% confluence, begin the differentiation process; aspirate off any growth medium and replace it with 500 &#181;L of differentiation medium (Table 1) containing 3.3 nM bone morphogenetic protein (BMP4) in each well.</li>
	<li>After 48 h, replace the medium with the differentiation medium (500 &#181;L per well) and the differentiation cocktail (Table 1).</li>
	<li>Remove the medium 72 h later and add 100 nM of insulin in 500 &#181;L of fresh differentiation medium. 
	NOTE: Cells begin to show a round appearance with small lipid droplets inside.</li>
	<li>Aspirate off any differentiation medium and replace it with 500 &#181;L of simple medium-2% FBS (Table 1) 48 h later. Induce insulin resistance with 4 ng/mL of TNF-&#945;. Include control wells without TNF-&#945; treatment.</li>
	<li>After 24 h of incubation, add 4 ng/mL TNF-&#945; in simple medium-0% FBS (Table 1). Maintain control wells without TNF-&#945; treatment.</li>
	<li>Activate the insulin signaling pathway 24 h later by adding 100 nM insulin and incubate for 15 min at 37 &#176;C in a 5% CO2 atmosphere. Remove the medium and wash with 500 &#181;L of PBS. Include control wells without insulin treatment.</li>
</ol>
3. Evaluation of insulin signaling pathway by western blot
<ol>
	<li>Protein extraction and quantification
	<ol>
		<li>Wash each well of cells with 500 &#181;L of PBS and keep on ice to lyse the cells in 50 &#181;L of RIPA buffer (Table 1) containing 1% protease inhibitor cocktail added just prior to sample isolation. Scrape the entire surface of the well with a tip and transfer the lysate to a 0.6 mL microcentrifuge tube (keep on ice).</li>
		<li>Incubate for 1 h at 4 &#176;C in a rotating shaker,&#160;mix by vortexing, and centrifuge at 8,000 &#215; g for 15 min at 4 &#176;C and recover the supernatant (keep on ice).</li>
		<li>Determine the protein concentration using the Bradford assay (do not dilute the sample to quantify).</li>
		<li>Take the necessary volume corresponding to 40-60 &#181;g and denature with 6x Laemmli buffer (Table 1) by&#160;incubating at 97 &#176;C for 15 min while shaking at 550 rpm. Freeze until use.</li>
	</ol>
	</li>
	<li>SDS-polyacrylamide gel electrophoresis
	<ol>
		<li>Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) with a 7.5% polyacrylamide gel of 1.5 mm thickness. Insert the gel into the tank and fill with running buffer (Table 1).</li>
		<li>Add 3 &#181;L of prestained protein standard to the first well of the gel and load each sample into subsequent wells.</li>
		<li>Run the gel at 80 V for approximately 15 min to ensure the sample enters the polyacrylamide concentrator gel matrix. Thereafter, increase the voltage to 90 V for 2.5 h or until the 37 kDa molecular weight marker can be seen at the bottom edge of the gel.</li>
		<li>Transfer the proteins from the gel to a nitrocellulose membrane in a semi-dry chamber for 35 min at 25 V. Beforehand, submerge the gel, membrane, and filters in transfer buffer (Table 1) for a few minutes.</li>
	</ol>
	</li>
	<li>Western blot
	<ol>
		<li>After transfer, wash the membrane with Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T 0.1%) (Table 1) for 3 min with shaking at 30 rpm.</li>
		<li>Block the membrane with blocking solution (Table 1) at 4 &#176;C for 1 h in constant rotation.</li>
		<li>Cut the membrane into three parts according to the molecular weight marker to incubate overnight with different primary antibodies (diluted in blocking solution) at 4 &#176;C in constant rotation: Phospho-IRS1 (Tyr608) antibody (1:1,000), expected band at 180 kDa; Phospho-Insulin receptor &#946; (Tyr1150/1151) antibody (1:1,000), expected band at 95 kDa; Phospho-Akt (Ser473) antibody (1:1,000), expected band at 60 kDa.</li>
		<li>Wash the membranes 5x for 5 min each with TBS-T 0.1%.</li>
		<li>Incubate the membranes with the corresponding peroxidase-coupled secondary antibody (diluted in blocking solution) for 2 h at room temperature in constant rotation: peroxidase affiniPure donkey anti-rabbit IgG (H+L) (1:5,000).</li>
		<li>Wash the membranes 5x for 5 min each with TBS-T 0.1%.</li>
		<li>Detect the proteins using the chemiluminescence detection system. Prepare the substrate according to the manufacturer&#39;s instructions.</li>
		<li>Place the blot in a plastic bag and add 200 &#181;L of chemiluminescent substrate to completely cover the membrane. Drain excess substrate and remove any air bubbles.</li>
		<li>Expose each blot for 30-60 s in a high-performance imaging system compatible with chemiluminescence.</li>
		<li>Strip the membranes (steps 10-13 of the western blot section) to break the antibody-antigen interactions and thus allow nitrocellulose membranes to be re-blotted. Recover the membranes and wash 3x for 5 min each with TBS-T 1% at 50 rpm.</li>
		<li>Incubate for 7 min with 10 mL of 0.2 M NaOH at 50 rpm.</li>
		<li>Wash 3x for 5 min each with tap water at 50 rpm.</li>
		<li>Wash for 10 min with TBS-T 1% at 50 rpm.</li>
		<li>Repeat steps 2-9 of the western blot section to incubate the membrane with anti-beta tubulin (55 kDa) as a loading control using 1:1,000 dilution.</li>
		<li>Perform densitometric analysis25&#160;for each protein using ImageJ software (https://imagej.nih.gov/).</li>
	</ol>
	</li>
</ol>

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R., Van Parijs, L., Lodish, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=F.Tumor+necrosis+factor-a+suppresses+adipocyte-specific+genes+and+activatesexpression+of+preadipocyte+genes+in+3T3-L1+adipocytes:+nuclear+factor-kappaB+activation+by+TNF-a+is+obligatory.">F.Tumor necrosis factor-a suppresses adipocyte-specific genes and activatesexpression of preadipocyte genes in 3T3-L1 adipocytes: nuclear factor-kappaB activation by TNF-a is obligatory.</a> Diabetes. 51 (5), 1319-1336 (2002).</li><li>Jager, J., Gre &#769;meaux, T., Cormont, M., Le Marchand-Brustel, Y., Tanti, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1b-induced+insulin+resistance+in+adipocytes+throughdown-regulation+of+insulin+receptor+substrate-1+expression.">Interleukin-1b-induced insulin resistance in adipocytes throughdown-regulation of insulin receptor substrate-1 expression.</a> Endocrinology. 148 (1), 241-251 (2007).</li><li>Rotter, V., Nagaev, I., Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-6+(IL-6)+induces+insulinresistance+in+3T3-L1+adipocytes+and+is,+like+IL-8+and+tumor+necrosis+factor-a,overexpressed+in+human+fat+cells+from+insulin-resistant+subjects.">Interleukin-6 (IL-6) induces insulinresistance in 3T3-L1 adipocytes and is, like IL-8 and tumor necrosis factor-a,overexpressed in human fat cells from insulin-resistant subjects.</a> The Journal of Biological Chemistry. 278 (46), 45777-45784 (2003).</li><li>Isidor, M. 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=Insulin+resistance+rewires+the+metabolic+gene+program+and+glucose+utilization+in+human+white+adipocytes.">Insulin resistance rewires the metabolic gene program and glucose utilization in human white adipocytes.</a> International Journal of Obesity. 46 (3), 535-543 (2021).</li><li>Houstis, N., Rosen, E. D., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+have+a+causal+role+in+multiple+forms+of+insulin+resistance.">Reactive oxygen species have a causal role in multiple forms of insulin resistance.</a> Nature. 440 (7086), 944-948 (2006).</li></ol>

The authors declare no conflicts of interest.

This paper provides a method for studying insulin resistance that uses primary adipocytes in culture treated with TNF-&#945;. This model has the advantage that primary adipocytes can be cultured under defined conditions for long periods of time with a tight control of cellular environmental factors26. The assay duration is 15-20 days, although variations in the percentage of differentiated adipocytes can occur between experiments. Primary adipocytes have advantages over cell lines since they have ...

Insulin is an anabolic hormone produced by pancreatic islet &#946;-cells and the key regulator of glucose and lipid metabolism. Among its many functions, insulin regulates glucose uptake, glycogen synthesis, gluconeogenesis, protein synthesis, lipogenesis, and lipolysis1. The initial molecular signal after insulin interaction with its receptor (IR) is the activation of the intrinsic tyrosine protein kinase activity of IR2, resulting in its autophosphorylation3&#160;and the subsequent activation of a family of proteins known as insulin receptor substrates (IRS), which binds to adaptor proteins leading to activation of a cascade of protein kinases4. Insulin activates two main signaling pathways: phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)&#160;and Ras-mitogen-activated protein kinase (MAPK). The former constitutes a major branch point or node4,5&#160;for the activation of numerous downstream effectors involved in diverse physiological functions, including the regulation of fuel homeostasis, whereas the latter regulates cell growth and differentiation4,6. Insulin actions ultimately depend on the cell type and physiological context7.
One of the main insulin-responsive metabolic tissues is the adipose tissue. White adipose tissue is the most abundant type of fat in humans and rodents, distributed within subcutaneous fat (between the skin and muscles) and visceral fat (around the organs in the abdominal cavity). Given their large volume, adipocytes or fat cells are the most abundant cell type in adipose tissue. These fat cells can be brown/beige (thermogenic), pink (in the mammary gland), and white8,9. White adipocytes keep the main energy reserves in the body in the form of triglycerides, an insulin-dependent process. Insulin promotes glucose transport and lipogenesis, while it inhibits lipolysis or lipid breakdown7,10. It also facilitates the differentiation of preadipocytes into adipocytes&#160;&#8212; the mature fat-storing cells11.
Insulin resistance occurs when a normal insulin level produces an attenuated biological response, resulting in compensatory hyperinsulinemia12. Insulin resistance is a condition associated with overweight and obesity5, that when combined leads to type 2 diabetes (T2D) and other metabolic diseases13. Hyperinsulinemia compensates insulin resistance in peripheral tissues to maintain normal blood glucose levels14. However, eventual &#946;-cell loss or exhaustion, together with exacerbated insulin resistance, leads to elevated blood glucose levels consistent with T2D5. Therefore, insulin resistance and hyperinsulinemia can contribute to the development of obesity-derived metabolic diseases15. Furthermore, obesity may cause chronic low-grade local inflammation promoting insulin resistance in adipose tissue15,16,17. In addition, obesity-derived alterations in adipose tissue, such as fibrosis, inflammation, and reduced angiogenesis and adipogenesis, lead to lower adiponectin serum levels (an insulin sensitizer) and increased secretion of factors such as plasminogen activator inhibitor 1 (PAI-1), free fatty acids, and exosomes into the bloodstream, exacerbating insulin resistance17.
Many aspects underlying insulin resistance remain unknown. In vitro and in vivo models have been developed to study the mechanisms mediating insulin resistance in major target tissues, including adipose tissue. The advantage of in vitro models is that researchers have more control of the environmental conditions and can evaluate insulin resistance in specific cell types. Particularly, adipocyte precursor cells (APCs) have the individual phenotype of the donor tissue, which might reflect physiology better than adipocyte cell lines. A main factor inducing insulin resistance in vitro is tumor necrosis factor-&#945; (TNF-&#945;). TNF-&#945; is a proinflammatory cytokine secreted by adipocytes and macrophages in adipose tissue18. While it is required for proper adipose tissue remodeling and expansion19, long-term exposure to TNF-&#945; induces insulin resistance in adipose tissue in vivo and in adipocytes in vitro20. Chronic TNF-&#945; treatment of several cell types leads to increased serine phosphorylation of both IR and IRS-1, thereby promoting decreased tyrosine phosphorylation21. Increased phosphorylation of IRS-1 on serine residues inhibits the IR tyrosine kinase activity and may be one of the key mechanisms by which chronic TNF-&#945; treatment impairs insulin action22,23. TNF-&#945; activates pathways involving the serine/threonine kinase inhibitor of nuclear factor &#312;B kinase &#946; (IKK&#946;) and c-Jun N terminal kinase (JNK)24. JNK induces a complex proinflammatory transcriptional program but also directly phosphorylates IRS-16.
Understanding the pathogenesis of insulin resistance has become increasingly important to guide the development of future therapies against T2D. APCs have proven to be an excellent model for the study of fat cell biology, including the sensitivity and resistance to insulin, and for identifying the intrinsic properties of adipocytes independent of the systemic environment. APCs can be easily obtained from different adipose depots, and under the appropriate conditions, differentiated into mature adipocytes. With this method, direct effects on insulin resistance/sensitivity can be evaluated in adipocytes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1. Isolation mouse adipocyte precursor cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACK lysing buffer&#160;</td>
			<td>LONZA</td>
			<td>10-548E</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Biotin Microbeads&#160;</td>
			<td>Miltenyi</td>
			<td>130-090-485</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CD31</td>
			<td>eBioscience</td>
			<td>13-0311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoMACS Pro Separator</td>
			<td>Miltenyi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix (matrigel)</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Sigma</td>
			<td>F0291</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Equitech-Bio, Inc.</td>
			<td>BAC63-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 Monoclonal Antibody (30-F11) - Biotin&#160;</td>
			<td>eBioscience</td>
			<td>13-0451-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1</td>
			<td>Worthington Biochem</td>
			<td>LS004197</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D1756</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>12800017</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM low glucose</td>
			<td>GIBCO</td>
			<td>31600-034</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>315-09</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>GIBCO</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS mix</td>
			<td>Sigma</td>
			<td>I3146</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid 2-phosphate</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>Millipore</td>
			<td>ESG1107</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>Linoleic acid-albumin</td>
			<td>Sigma</td>
			<td>L9530</td>
			<td></td>
		</tr>
		<tr>
			<td>MCDB 201 medium</td>
			<td>Sigma</td>
			<td>M6770</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin</td>
			<td>InvivoGen</td>
			<td>ant-nr-2</td>
			<td></td>
		</tr>
		<tr>
			<td>PDGF-BB&#160;</td>
			<td>Peprotech</td>
			<td>315-18</td>
			<td>Growth factor</td>
		</tr>
		<tr>
			<td>Peniciline-Streptomycine</td>
			<td>BioWest</td>
			<td>L0022-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-Separation Filters (70 &#181;m)</td>
			<td>Miltenyi</td>
			<td>130-095-823</td>
			<td></td>
		</tr>
		<tr>
			<td>Purified Rat Anti-Mouse CD16 / CD32&#160;</td>
			<td>BD Pharmingen</td>
			<td>553142</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA&#160;</td>
			<td>GIBCO</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>2. Adipocyte differentiation and insulin resistance induction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine [IBMX]</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td>Differentiation cocktail</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>R&#38;D Systems</td>
			<td>5020-BP-010</td>
			<td>Differentiation cocktail</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D1756</td>
			<td>Differentiation cocktail</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Cayman</td>
			<td>71742</td>
			<td>Differentiation cocktail</td>
		</tr>
		<tr>
			<td>TNF&#945;</td>
			<td>R&#38;D Systems</td>
			<td>210-TA-005&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>3. Evaluation of insulin signaling pathway by western blot</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-beta tubulin antibody</td>
			<td>Abcam</td>
			<td>ab6046</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>BioRad</td>
			<td>161-0404</td>
			<td>Laemmli buffer</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>RIPA buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>E4378</td>
			<td>RIPA buffer</td>
		</tr>
		<tr>
			<td>FluorChem E system</td>
			<td>ProteinSimple</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G6279</td>
			<td>Laemmli buffer</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma</td>
			<td>G7126</td>
			<td>Running and Transfer buffer</td>
		</tr>
		<tr>
			<td>Igepal</td>
			<td>Sigma</td>
			<td>I3021</td>
			<td>RIPA buffer</td>
		</tr>
		<tr>
			<td>2- mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148</td>
			<td>Laemmli buffer</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>JT Baker</td>
			<td>907007</td>
			<td>Transfer buffer</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>JT Baker</td>
			<td>3624-05</td>
			<td>TBS-T</td>
		</tr>
		<tr>
			<td>NaF</td>
			<td>Sigma</td>
			<td>77F-0379</td>
			<td>RIPA buffer</td>
		</tr>
		<tr>
			<td>NaOH&#160;</td>
			<td>JT Baker</td>
			<td>3722-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Na4P2O7</td>
			<td>Sigma</td>
			<td>114F-0762</td>
			<td>RIPA buffer</td>
		</tr>
		<tr>
			<td>Na3VO4</td>
			<td>Sigma</td>
			<td>S6508</td>
			<td>RIPA buffer</td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane&#160;</td>
			<td>BioRad</td>
			<td>1620112</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonfat dry milk</td>
			<td>BioRad</td>
			<td>1706404</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Prestained protein standard&#160;</td>
			<td>BioRad</td>
			<td>1610395</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail&#160;</td>
			<td>Sigma</td>
			<td>P8340-5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase AffiniPure Donkey Anti-Rabbit IgG (H+L)&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-035-132</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho- Insulin Receptor &#946;&#160;</td>
			<td>Cell signaling</td>
			<td>3024</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Akt (Ser473) Antibody</td>
			<td>Cell signaling</td>
			<td>9271</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-IRS1 (Tyr608) antibody</td>
			<td>Millipore</td>
			<td>9432</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharose&#160;</td>
			<td>JT Baker</td>
			<td>407205</td>
			<td>RIPA buffer</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>BioRad</td>
			<td>1610302</td>
			<td>Running and laemmli buffer</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34577</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Promega</td>
			<td>H5135</td>
			<td>Running, transfer and laemmli buffer</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>JT Baker</td>
			<td>4103-02</td>
			<td>RIPA buffer - TBS</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td>TBS-T</td>
		</tr>
	</tbody>
</table>

differentiated mouse adipocytes in primary culture: a model of insulin resistance

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-&#945; (TNF-&#945;).
Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-&#945;, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

Over the last few years, the increased prevalence of obesity and T2D has prompted an intense search for the mechanisms mediating insulin resistance in adipose tissue. With the protocol described here, APCs can be differentiated into mature adipocytes to evaluate insulin resistance and sensitivity. Once the APCs reach confluence, it takes 10 days to complete their differentiation into mature adipocytes and their TNF-&#945;-mediated induction of insulin resistance (Figure 1).
<p class="jov...

Watch this Scientific Journal Video about Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance at JoVE.com

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance ...

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2.3K Views.  Universidad Nacional Autónoma de México (UNAM). This protocol describes the isolation of mouse preadipocytes from subcutaneous fat, their differentiation into mature adipocytes, and the induction of insulin resistance. Insulin action is evaluated by the phosphorylation/activation of members of the insulin signaling pathway through western blot. This method allows direct determination of insulin resistance/sensitivity in primary adipocytes. 

Video: Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

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 ...

Validation of a Mouse Model to Disrupt LINC Complexes in a Cell-specific Manner

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the Nucleoskeleton and Cytoskeleton (LINC complexes). These protein scaffolds span the nuclear envelope and connect the interior of the nucleus to components of the surrounding cytoplasmic cytoskeleton. LINC complexes consist of two evolutionary-conserved protein families, Sun proteins and Nesprins that harbor C-terminal molecular signature motifs called the SUN and KASH domains, respectively. Sun proteins are transmembrane proteins of the inner nuclear membrane whose N-terminal nucleoplasmic domain interacts with the nuclear lamina while their C-terminal SUN domains protrudes into the perinuclear space and interacts with the KASH domain of Nesprins. Canonical Nesprin isoforms have a variable sized N-terminus that projects into the cytoplasm and interacts with components of the cytoskeleton. This protocol describes the validation of a dominant-negative transgenic mouse strategy that disrupts endogenous SUN/KASH interactions in a cell-type specific manner. Our approach is based on the Cre/Lox system that bypasses many drawbacks such as perinatal lethality and cell nonautonomous phenotypes that are associated with germline models of LINC complex inactivation. For this reason, this model provides a useful tool to understand the role of LINC complexes during development and homeostasis in a wide array of tissues.

Nuclear envelope proteins play a central role in many basic biological processes and have been implicated in a variety of human diseases. This protocol describes a new Cre/Lox-based mouse model that allows for the spatiotemporal control of LINC complexes disruption.

Nuclear migration and anchorage within developing and adult tissues relies heavily upon large macromolecular protein assemblies called LInkers of the ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...