The authors are grateful to Cristina Godio at Centro Nacional de Biotecnolog&#237;a in Madrid, Spain, Mari Gantner at The Scripps Research Institute, La Jolla, and Anastasia Kralli at Johns Hopkins University, Baltimore, for assistance optimizing this protocol based on the initial work of Kahn et al.18. This work was funded by NIH grants DK114785 and DK121196 to E.S.

This protocol follows all IACUC guidelines of The Scripps Research Institute and the University of Wisconsin &#8211; Madison School of Medicine and Public Health.
1. Collection and digestion of adipose depots (day 1)
<ol>
	<li>Prepare two 1.5 mL tubes for each pup: one for brown adipose tissue (BAT) and one for white adipose tissue (WAT). Add 250 &#181;L of phosphate-buffered saline (PBS) + 200 &#181;L of 2x isolation buffer (123 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 5 mM glucose, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), penicillin-streptomycin, and 4% fatty acid-free bovine serum albumin) to each tube. Keep solutions sterile and on ice.</li>
	<li>Place the pups into small chambers (e.g., one well of a 6-well plate), and set them on ice until they become hypothermic. Make sure that there is no direct contact between the pups and the ice. Prick a paw with a tip to assure loss of consciousness, and euthanize the pups by decapitation using sharp scissors. 
	NOTE: if working with different genotypes, prepare an additional tube to collect a tail biopsy (3 mm cut) for genotyping. If genotyping is performed immediately, keep the euthanized pups on ice until the dissection to collect adipose depots.</li>
	<li>Calculate and weigh out the amount of collagenase needed to digest all the depots. Add 50 &#181;L of 15 mg/mL collagenase type I in 2x isolation buffer to each tube. Do not resuspend the collagenase in isolation buffer until all the tissues have been collected.</li>
	<li>To collect subcutaneous WAT, cut the skin around the pup&#39;s abdomen (avoid peritoneal rupture), and gently pull the skin down below the legs. Without taking the skin, carefully collect the fat depot, which will appear as a clear (P1 or younger) or white (P2 and older), thin, elongated tissue attached on the inside or on top of the quadriceps (Figure 1B). Rinse the fat depot in PBS, and place it in one of the tubes containing 250 &#181;L of PBS + 200 &#181;L of 2x isolation buffer. Keep on ice. 
	NOTE: P0 and P1 mice give the best yield. In P0-1 pups, the subcutaneous WAT depot is nearly transparent. In P2 mice and older, the depot is easier to identify because it is already turning white, indicating the presence of fully mature, lipid-loaded, white adipocytes.</li>
	<li>To collect interscapular BAT, pull the skin from the shoulder blades over the head. Lift the BAT - the deep red tissue between the shoulder blades - and carefully make incisions all around it to detach it from the body (Figure 1B). Check for consistency and color; rinse with PBS; place it in the other tube containing 250 &#181;L of PBS + 200 &#181;L of 2x isolation buffer; and keep on ice. 
	NOTE: When harvesting BAT from P2 mice and older, carefully remove the white adipose tissue surrounding the BAT. It consists of a thin, soft, white sheet of adipocytes located between the skin and the BAT, which is deeper between the shoulder blades.</li>
	<li>Once all depots have been collected, gently mince each depot (4-6 times) using small scissors directly inside the tubes.</li>
	<li>Resuspend collagenase type I in the appropriate volume of 2x isolation buffer to obtain a 10x stock solution at 15 mg/mL. Add 50 &#181;L of 10x collagenase to each tube, keeping the tubes on ice until collagenase has been added to all the tubes.</li>
	<li>Invert the tubes quickly to mix, and transfer to a temperature-controlled mixer. Incubate the samples for 30 min at 37 &#176;C on a shaker (1,400 rpm frequency for effective sample mixing).</li>
</ol>
2. Plating preadipocytes (day 1)
<ol>
	<li>After digesting the tissues, place the tubes back on ice. 
	NOTE: From this step onwards, all work is carried out in sterile conditions in a biosafety cabinet.</li>
	<li>Strain the digested tissues through a 100 &#181;m cell strainer into new 50 mL tubes. If working only with WT mice, or if genotypes are known, pool the relevant, dissociated BATs together; repeat this for the WATs. To maximize the cell yield, rinse each tube with 1 mL of isolation medium (Dulbecco&#39;s modified Eagle medium (DMEM) + 20% fetal bovine serum (FBS), 10 mM HEPES, 1% penicillin-streptomycin), and filter it through the cell strainer. If working with unknown genotypes, go to step 2.4 
	NOTE: FBS is an essential determinant of both preadipocyte proliferation and differentiation. Rigorously test different lots of FBS to ensure high performance and consistency between lots.</li>
	<li>Dilute BAT and WAT suspensions to plate 2-3 wells of a 6-well plate for each BAT sample and 4-6 wells of a 6-well plate for each WAT sample. For instance, if 6 BATs and 6 WATs were pooled together, dilute the BAT cell suspension up to 24-36 mL and the WAT cell suspension up to 48-72 mL. Plate 2 mL of the cell suspensions per well.</li>
	<li>For samples with unknown genotypes, keep each sample separate; place a 100 &#181;m cell strainer on top of each well of a 6-well plate, and filter one sample per well. Rinse the tube with 1.5 mL of isolation medium, and pass it through the strainer, making up the final volume to 2 mL per well. Discard the cell strainer, place the lid on the plate, and transfer the plate to the incubator. 
	NOTE: The medium in the wells should look turbid&#160;due to floating blood cells, cell debris, and lipids from lysed cells.</li>
	<li>1-1.5 h after plating, aspirate media&#160;and wash with 2 mL of DMEM without serum. Gently agitate the plate to detach blood cells from the bottom of the wells. After 3 washes, add 2 mL of fresh isolation medium, and transfer the cells to the incubator (37 &#176;C, 5% CO2). 
	NOTE: Check the cells under the microscope. Floating material (blood cells, cellular debris) should be minimal. Brown preadipocytes will appear as small non-translucent cells, whereas white preadipocytes will display a more elongated form. Both brown and white preadipocytes should be tightly attached to the plate.</li>
</ol>
3. Expansion of preadipocyte culture (day 2 to day 5)
<ol>
	<li>On the next day, aspirate the medium, wash the cells with 2 mL of DMEM without serum, and add back 2 mL of the isolation medium.</li>
	<li>Repeat step 3.1 once every 2 days until the cells reach 80-90% confluence. 
	NOTE: For primary brown preadipocytes, reaching 80-90% confluence can take 4-5 days. For primary white preadipocytes, it usually takes 2-3 days. Once the preadipocytes reach 60% confluence, they can be efficiently infected with viral particles for knockdown or overexpression experiments. Infect preadipocytes with an appropriate viral load for up to 8 h. The use of cationic polymers to increase infection efficiency is discouraged, as it often results in toxicity and in a significant reduction of the adipogenic potential of infected preadipocytes.</li>
	<li>To split the cells, coat new destination plates using a sterile solution of 0.1% w/v gelatin (dissolved in distilled water; do not use any heat). Use enough volume to cover the bottom of the well/dish. Incubate plates at 37 &#176;C until the cells are ready to be seeded (at least 10 min). 
	NOTE: This step is optional. Coating of cell culture plates does not affect the yield or differentiation potential, but it substantially simplifies the maintenance and handling of differentiating adipocytes.</li>
	<li>When cells reach sub-confluent density (85-95%), aspirate the medium, wash with PBS, and add trypsin for 3 min to detach the cells. Block trypsin activity by adding 2.5x trypsin volume of isolation medium. Pipette the cell suspension up and down to maximize cell recovery, and transfer into a new tube. Wash the wells with 1 mL of isolation medium, and add it to the first collection.</li>
	<li>Centrifuge the cells for 5 min at 800-1200 &#215; g, aspirate the supernatant, and resuspend the cells in 3-5 mL of the isolation medium. Count the cells, and dilute them to the desired final density. 
	NOTE: For instance, 50,000-80,000 cells/mL (2.5, 1, and 0.5 mL for 6-, 12- and 24-well plates, respectively) will result in fully confluent wells within 72-96 h.</li>
	<li>Aspirate the coating solution in step 3.3, and wash off excess gelatin with PBS. Seed the cells, and return the plates to the incubator until fully confluent.</li>
</ol>
4. Differentiation of white and brown preadipocytes (days 7 - 12)
<ol>
	<li>When preadipocytes become confluent, aspirate the medium, and replace it with differentiation medium consisting of 10% FBS in DMEM containing 170 nM insulin, 1 &#181;M dexamethasone, and 0.5 mM 3-isobuthyl-1-methylxantine. If differentiating BAT preadipocytes, also add 1 nM triiodothyronine (T3). Mark the day on which adipogenic differentiation is induced as day 0 of differentiation. 
	NOTE: Both white and brown preadipocytes can spontaneously differentiate upon reaching confluence. However, to maximize adipocyte differentiation, the traditional pro-adipogenic cocktail described above (plus T3 for BAT preadipocytes) should be used.</li>
	<li>After 48 h (day 2 of differentiation), refresh the medium with maintenance medium consisting of 10% FBS in DMEM and 170 nM insulin (plus 1 nM T3 for BAT). 
	NOTE: On day 2, small lipid droplets accumulating in the cells under the microscope can be observed using standard bright field.</li>
	<li>Repeat step 4.2 once every 2 days until cells are used for experiments. 
	NOTE: On day 4 or 5 of differentiation, the majority of cells will appear loaded with lipids. Terminally differentiating adipocytes can be kept in culture longer or used at this stage for experiments.</li>
</ol>
5. Re-plating for bioenergetics experiments
NOTE: If the intention is to perform bioenergetics studies in mature adipocytes in the 96-well format, the following steps need to be taken. Ideally, cells that have just fully differentiated (day 4 or 5) should be used. The procedure described below starts from 1 well of a 6-well plate.
<ol>
	<li>Prior to trypsin digestion, prepare coating for 96-well plates. Add 50 &#181;L of 0.1% gelatin to each well. Leave the plate in a tissue culture incubator until the cells are ready to be seeded.</li>
	<li>On day 4 or 5 of differentiation, aspirate the medium, wash with 1 mL of PBS, and add 300 &#181;L of 0.25% trypsin-ethylenediamine tetraacetic acid (for 1 well of a 6-well plate). Gently tilt the plate to ensure trypsin completely covers the bottom of the well; incubate for 2 min at room temperature. Block the action of trypsin with 700 &#181;L of maintenance medium, pipette up and down to maximize cell recovery, and transfer the cell suspension into a new 1.5 mL tube.</li>
	<li>Centrifuge the cells at 1200 &#215; g for 5 min. Remove the supernatant, resuspend the pellet in 1 mL of maintenance medium, and count the cells. 
	NOTE: One well of a 6-well plate should yield approximately 1.5-1.8 million cells.</li>
	<li>Dilute the cells to have a final concentration of 60,000 to 80,000 cells/mL for brown adipocytes and 100,000 to 120,000 cells/mL for white adipocytes. Plate 100 &#181;L of the cell suspension per well in gelatin-coated 96-well plates. 
	NOTE: One well of a 6-well plate provides enough cells for up to two 96-well plates. For bioenergetics experiments, low-density plating (30-40% confluence) is needed to enable the accurate measurement of oxygen consumption and avoid oxygen depletion in the wells during the assay.</li>
	<li>Allow the cells to adhere to the bottom of the plate for at least 48 h. If cells are kept in the plate for more than 48 h, refresh the medium after 2 days.</li>
	<li>On the day of the assay, aspirate the medium, and replace it with assay medium. Incubate for 1 h at 37 &#176;C in a non-CO2-controlled incubator, and perform the assay as per the manufacturer&#39;s instructions.</li>
</ol>

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The authors have nothing to disclose.

Adipose tissue is critical for systemic insulin sensitivity and glucose homeostasis20. Obesity-linked adipocyte dysfunction is tightly associated with the onset of type 2 diabetes. Therefore, greater understanding of the basic biology and physiology of adipose tissue may enable the design of new treatments for metabolic disorders. As a complement to direct functional and transcriptional analysis of mature adipocytes isolated from fat depots21,22, cultured primary adipocytes have been shown to recapitulate many aspects of adipose tissue pathophysiology, including secretion of adipokines, resistance to insulin in response to pro-inflammatory stimuli, and induction of the thermogenic program23,24,25. Although previous protocols have described the isolation of adipocyte precursors from adult mice11,12, this protocol provides a method for the efficient isolation of cells from newborn pups. This strategy yields a significantly larger population of white and brown adipocyte progenitors with higher differentiation potential, as postnatal adipose depots are still relatively undifferentiated compared to the adipose depots of adult mice26. Moreover, cells isolated using this method are already committed and yield fully functional differentiated adipocytes that express markers of mature cells and exhibit their unique physiological features, including lipid storage and thermogenic capacity.
Primary cells cannot be expanded indefinitely in vitro. In addition, primary preadipocytes start to lose their adipogenic potential after several passages in culture. This is likely because continual cell culture results in an intrinsic enrichment of less committed, more proliferative cells. Thus, one limitation of this protocol is the window of time during which preadipocytes can be used. Adipogenic potential is fully preserved if cells are induced to differentiate within 7-8 days from the time of isolation. Adipocyte progenitors are particularly resistant to enzymatic digestion, but it is nonetheless important to correctly time the collagenase treatment. Overdigestion of tissues may result in reduced cell survival and decreased ability of cells to adhere to the plate. Throughout the expansion phase, both white and brown preadipocytes are strongly adherent and can tolerate vigorous washes and frequent media exchange. However, the use of coated plates is recommended when preadipocytes are induced to differentiate. Mature, lipid-laden adipocytes have decreased surface adhesion and cell-cell interactions, resulting in a tendency to detach during differentiation unless gently handled. The most delicate step of the protocol is the induction of differentiation. FBS, insulin, T3, and other drugs must be tested, and their final concentrations optimized, to obtain the highest extent of differentiation. A PPAR&#947; agonist (e.g., rosiglitazone) can also be added to further stimulate adipogenesis.
It is important to note that the differentiation conditions can be adapted to the experimental needs. For instance, in screens with genetic or chemical libraries designed to identify proteins/compounds that enhance white and/or brown adipocyte differentiation, preadipocytes can be induced to differentiate using a minimal permissive medium that includes 10% FBS in DMEM and 170 nM insulin only. Assessment of each component of the differentiation cocktail is recommended to determine ideal assay windows for differentiation assays. T3 and dexamethasone are dispensable for primary white and brown adipocyte differentiation. These conditions will ensure a low rate of differentiation in control cells, thus increasing the window of the assay and maximizing the ability to detect pro-adipogenic factors. Cultures of primary brown and white adipocytes are a powerful tool to interrogate adipocyte cell-autonomous function in response to genetic manipulation and metabolic stresses to complement the study of brown and white adipose tissue in vivo. Hence, protocols for isolation and culture of primary preadipocytes are needed to enable reproducible, high-throughput investigations of adipocyte function in vitro. The strategy described here allows study of primary white and brown preadipocytes that can be differentiated into fully mature adipocytes and tested under a variety of experimental manipulations.

Obesity results from a chronic imbalance between energy intake and energy expenditure. As obesity develops, white adipocytes undergo a massive expansion in cell size that results in hypoxia in the microenvironment, cell death, inflammation, and insulin resistance1. Dysfunctional, hypertrophied adipocytes cannot properly store excess lipids, which accumulate instead in other tissues where they dampen insulin action2,3. Agents that improve adipocyte function and restore normal lipid partitioning amongst tissues are predicted to be beneficial for the treatment of obesity-associated conditions characterized by insulin resistance such as type 2 diabetes. Phenotypic screens in adipocytes using immortalized cell lines, such as 3T3-L1, F442A, and 10T &#189;, have proven useful to identify genetic factors that regulate adipogenesis and to isolate pro-adipogenic molecules with anti-diabetic properties4,5,6,7. These cell lines, however, do not fully reflect the heterogeneity of cell types present in adipose depots, which includes white, brown, beige, and other adipocyte subtypes with unique characteristics, all of which contribute to systemic homeostasis8,9,10. Further, cultured cell lines often show a diminished response to external stimuli.
In contrast, cultures of primary adipocytes recapitulate more accurately the complexity of in vivo adipogenesis, and primary adipocytes show robust functional responses. Primary preadipocytes are typically isolated from the stromal vascular fraction of adipose depots of adult mice11,12,13,14. However, because the adipose depots of adult animals consist primarily of fully mature adipocytes that have a very slow turnover rate15,16,17, this approach yields a limited quantity of preadipocytes with a low proliferation rate. Therefore, isolation of preadipocytes from newborn mice is preferable to obtain large quantities of rapidly growing cells that can be differentiated in vitro. Here, a protocol has been described, inspired by the initial work with primary brown adipocytes of Kahn et al.18 to efficiently isolate both white and brown preadipocytes that can be expanded and differentiated in vitro into fully functional primary adipocytes (Figure 1A). The advantage of isolating primary cells from newborn, as opposed to adult mice, is that the adipose depots are rapidly growing and are thus a rich source of actively proliferating preadipocytes17. Cells isolated using this protocol have high proliferative capacity, enabling rapid scale-up of cultures. In addition, preadipocytes from newborn pups display higher differentiation potential than adult progenitors, which reduces well-to-well variability in the extent of differentiation and thus increases reproducibility.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-Isobutyl-1-methylxanthine (IBMX)</td>
			<td>Sigma-Aldrich</td>
			<td>I7018</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>AdipoRed (Nile Red)</td>
			<td>Lonza</td>
			<td>PT-7009</td>
			<td></td>
		</tr>
		<tr>
			<td>Antimycin A</td>
			<td>Sigma-Aldrich</td>
			<td>A8674</td>
			<td></td>
		</tr>
		<tr>
			<td>BenchMark Fetal Bovine Serum</td>
			<td>Gemini Bioproducts LLC</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase, Type 1&#160;&#160;</td>
			<td>Worthington Biochemical Corp</td>
			<td>LS004196</td>
			<td></td>
		</tr>
		<tr>
			<td>ddH2O</td>
			<td>Sigma-Aldrich</td>
			<td>6442</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma-Aldrich</td>
			<td>D5030</td>
			<td>For Bioenergetics studies</td>
		</tr>
		<tr>
			<td>DMEM, High Glucose, Glutamax</td>
			<td>Gibco</td>
			<td>10569010</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Fatty Acid-Free BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A8806</td>
			<td></td>
		</tr>
		<tr>
			<td>FCCP</td>
			<td>Sigma-Aldrich</td>
			<td>C2920</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>G1890</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich</td>
			<td>I6634</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Norepinephrine</td>
			<td>Cayman Chemical</td>
			<td>16673</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligomycin</td>
			<td>Sigma-Aldrich</td>
			<td>75351</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosiglitazone</td>
			<td>Sigma-Aldrich</td>
			<td>R2408</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>557368</td>
			<td></td>
		</tr>
		<tr>
			<td>Seahorse XFe96 FluxPak</td>
			<td>Agilent Technologies</td>
			<td>102416-100</td>
			<td>For Bioenergetics studies</td>
		</tr>
		<tr>
			<td>Surgical forceps</td>
			<td>ROBOZ Surgical Instrument Co</td>
			<td>RS-5158</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Scissors</td>
			<td>ROBOZ Surgical Instrument Co</td>
			<td>RS-5880</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Eppendorf</td>
			<td>T1317</td>
			<td></td>
		</tr>
		<tr>
			<td>triiodothyronine (T3)</td>
			<td>Sigma-Aldrich</td>
			<td>642511</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and differentiation of primary white and brown preadipocytes from newborn mice

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

Section 1 of the protocol will yield a heterogeneous suspension of cells that are visible under a standard light microscope. Filtering of digested tissues with a cell strainer (section 2) will remove undigested tissue. However, some cellular debris, blood cells, and mature adipocytes will pass through (Figure 1C). Gentle washes 1 h after plating will remove non-relevant cells as preadipocytes attach rapidly to the bottom of the well (Figure 1C). In section 3, adipocyte precursors are expanded to obtain the number of cells required for the experimental plan. Although both white and brown preadipocytes isolated from newborn pups have high proliferative capacity, the yield of white preadipocytes is generally twice that of brown preadipocytes on a per-depot basis (Figure 2A). Therefore, if synchronized cultures are desired, the starting density of white preadipocytes must be calculated accordingly. Section 4 offers guidelines to obtain fully mature adipocytes.
At the end of differentiation, cells will appear loaded with lipid droplets and express classical markers of white and brown adipocytes, respectively (Figure 2B,C). Both white and brown adipocytes can be used for bioenergetics studies as described in section 5. A comparative analysis of oxygen consumption in a mitochondrial stress test of primary white and brown adipocytes under basal conditions, as well as in response to known stimulators of mitochondrial function (e.g., norepinephrine) is shown in Figure 2D. Upon isolation, it is also possible to differentiate primary white and brown adipocytes on the plates that will be directly used for bioenergetic experiments19. In this case, preadipocytes are isolated and plated as described in sections 1 and 2. When cells become confluent, they are induced to differentiate as described in section 4 until they reach terminal differentiation and are ready for bioenergetics analysis. This procedure is common for a 24-well plate format, but less so for 96-well plates.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62005/62005fig1.jpg" /> 
Figure 1: Collection and processing of fat pads. (A) Schematic representation of primary white (top) and brown (bottom) adipocyte isolation. (B) Subcutaneous white (top) and brown (bottom) adipose depots. In P0 mice, subcutaneous WAT is almost invisible, but becomes distinguishable on ~day 2 after birth. In contrast, BAT has a distinct dark color even at P0. In older pups, the BAT is surrounded by a thin superficial layer of WAT, which requires removal when the tissue is dissected. (C) Representative images of primary white and brown precursor cells after filtration through the 100 &#181;m cell strainer, after the initial washes, and 24 h after isolation. Scale bars = 100 &#181;m. Abbreviations: WAT = white adipose tissue; BAT = brown adipose tissue. <a href="https://www.jove.com/files/ftp_upload/62005/62005fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62005/62005fig2.jpg" /> 
Figure 2: Differentiation of adipocyte progenitors. (A) Average number of subcutaneous WAT and BAT preadipocytes obtained per newborn (P0) pup. (B) Representative images of terminally differentiated white and brown adipocytes. For fluorescence imaging, cells were incubated with Nile Red and Hoechst 33342 to stain neutral lipids and nuclei, respectively. Scale bars = 100 &#181;m.(C) Gene expression analysis of classical adipocyte markers, white-beige markers, and brown-specific markers in primary white and brown adipocytes differentiated for 6 days (n=3). For each gene, BAT expression is relative to WAT levels (set to 1). (D) OCR of white and brown adipocytes in a mitochondrial stress test and in response to norepinephrine (n=3). Brown adipocytes show uncoupled respiration and a robust response to norepinephrine, whereas white adipocytes show little uncoupled respiration and no significant response to adrenergic stimulation. *p&#60;0.05; **p&#60;0.01, determined by two-tailed Student&#39;s t-test. Abbreviations: WAT = white adipose tissue; BAT = brown adipose tissue; OCR = oxygen consumption rate; Oligo = oligomycin; FCCP = carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; RAA = rotenone, antimycin A; PPAR&#947; = peroxisome proliferator-activated receptor gamma; Acrp30 = adipocyte complement-related protein of 30 kDa; Fabp4 = fatty acid-binding protein 4; Glut4 = glucose transporter 4; Cd36 = cluster of differentiation 36; Retn = resistin; Slc27a1 = solute carrier family 27 member 1; Ear2 = eosinophil-associated, ribonuclease A family 2; Pgc1a = PPAR&#947; coactivator 1alpha; Prdm16 = positive regulatory domain I-binding factor 1 (PRDI-BF1) and retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) homologous domain containing 16; Eva1 = epithelial V-like antigen 1; Cidea = cell death-inducing DNA fragmentation factor-alpha-like effector A; Ucp1 = uncoupling protein 1; Dio2 = type 2 deiodinase. <a href="https://www.jove.com/files/ftp_upload/62005/62005fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice at JoVE.com

Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white ...

isolation-differentiation-primary-white-brown-preadipocytes-from

Universidad San Sebastian

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JoVE Journal

Biology

11.2K Views.  The Scripps Research Institute, La Jolla, CA, USA. This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

Video: Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and memory. Primary hippocampal cell culturing allows neuroscientists to examine the activity and properties of neurons at the individual cell and single synapse level. In this video, we will demonstrate how to isolate and grow primary hippocampal cells from newborn rats. The hippocampus may be isolated from each newborn animal in as short as 2 to 3 minutes, and the cultures can be maintained for up to two weeks. We will also briefly demonstrate how to use these hippocampal neurons for ratiometric calcium imaging. While this protocol describes the process for the hippocampus, with little to no modification, it can be applied to other regions of the brain.

The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

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 ...

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, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Differentiation and Imaging of Brown Adipocytes from the Stromal Vascular Fraction of Interscapular Adipose Tissue from Newborn Mice

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with multiple lipid droplets, a central nucleus, a high mitochondrial content, and the expression of uncoupling protein 1 (UCP1). BAT has been proposed as a potential therapeutic target for obesity and its associated metabolic disorders due to its ability to dissipate metabolic energy as heat. To investigate BAT function and regulation, brown adipocyte culturing is indispensable. The present protocol optimizes tissue processing and cell differentiation for culturing brown adipocytes from newborn mice. Additionally, procedures for the imaging of differentiated adipocytes with both confocal immunofluorescence and transmission electron microscopy are shown. In the brown adipocytes differentiated with the techniques described herein, the major defining features of classical BAT are preserved, including high UCP1 levels, increased mitochondrial mass, and very close physical contact between the lipid droplets and mitochondria, making this method a valuable tool for BAT studies.

Preadipocytes are isolated from the stromal vascular fraction of interscapular brown adipose tissue from newborn mice and differentiated into cells that accumulate lipid droplets, express molecular markers, and show the mitochondrial morphology of mature brown adipocytes. These cells are further analyzed by immunofluorescence and transmission electron microscopy.

Brown adipose tissue (BAT) is only present in mammals and has a thermogenic function. Brown adipocytes are characterized by a multilocular cytoplasm with ...

Unveiling Inflammatory Arthritis&#58; Exploring Functions of Synovial Cells

Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central roles in the pathogenesis of rheumatoid arthritis. It is important to understand the functions of both cell populations to reveal the mechanisms underlying pathological progression and remission in inflammatory arthritis. In general, in vitro experimental conditions should mimic the in vivo environment as much as possible. Primary tissue-derived cells have been used in experiments characterizing synovial fibroblasts in arthritis. In contrast, in experiments investigating the biological functions of macrophages in inflammatory arthritis, cell lines, bone marrow-derived macrophages, and blood monocyte-derived macrophages have been used. However, it is unclear whether such macrophages actually reflect the functions of tissue-resident macrophages. To obtain resident macrophages, previous protocols were modified to isolate and expand both primary macrophages and fibroblasts from synovial tissue in an inflammatory arthritis mouse model. These primary synovial cells may be useful for in vitro analysis of inflammatory arthritis.

Author Spotlight: Isolation and Culture of Primary Synovial Macrophages and Fibroblasts from Murine Arthritis Tissue  

The present study provides a modified protocol to isolate synovial macrophages and fibroblasts from murine inflammatory arthritis tissue.

Rheumatoid arthritis is an autoimmune disease that leads to chronic inflammation of joints. Synovial macrophages and synovial fibroblasts have central ...