The authors wish to acknowledge grant support from 5 For The Fight and Huntsman Cancer Institute (KIH), the V Foundation for Cancer Research (KIH), and DK133455 (KIH). Schematics of the protocol created with Biorender. Additionally, the authors would like to thank Stephanie Giagnocavo and the Cell Imaging Core at the University of Utah for imaging help, and Mark Lee for feedback on the manuscript.

All animal experiments described were approved by the University of Utah&#39;s Committee on the Use and Care of Animals. This protocol has been optimized for the isolation of mature adipocytes from C57BL/6 male and female mice aged 8-16 weeks, with body weights ranging from 20-45 g. Details of the reagents and equipment used in this study are provided in the Table of Materials.
1. Solution preparation
<ol>
	<li>Prepare collagenase buffer. Add 100 mg of collagenase powder, 1 mL of 1 M HEPES buffer, 500 &#181;L of 100x P188, 50 &#181;L of 1 M CaCl2, 0.05 g of BSA, and 48.45 mL of Medium 199. Sterile filter the solution, aliquot, and store at -20 &#176;C.</li>
	<li>Make cell culture media. Combine 500 mL of DMEM, 50 mL of FBS, 5 mL of Penicillin-Streptomycin, and 5 mL of L-glutamine supplement.</li>
</ol>
2. Collection of white adipose tissue depots from mice
<ol>
	<li>Prepare a 1 x 10 cm plate with 200 &#181;L of HBSS for each different depot and condition as appropriate and place it on ice.</li>
	<li>Retrieve the animals from the animal house and euthanize the mice in accordance with the guidelines of the Institution&#39;s Animal Care and Use Committee. Here, animals were euthanized by cervical dislocation under deep anesthesia.</li>
	<li>Weigh and record the weight of each mouse if appropriate.</li>
	<li>Sterilize the mouse by spraying 70% ethanol to coat the exterior.</li>
	<li>Harvest the appropriate fat pads from the mouse18,19. 
	NOTE: Take care to remove the lymph nodes from the subcutaneous depot before placing the tissue into the dish.</li>
	<li>Weigh each fat and record the weight.</li>
	<li>Place the fat pad in the prepared 10 cm plate on ice.</li>
</ol>
3. Preparation of white adipose tissue explants
NOTE: This step is optional.
<ol>
	<li>At least 12 h before beginning dissection, thaw the basement membrane matrix (Matrigel) at 4 &#176;C on ice and chill one box of sterile 200 &#181;L pipette tips at 4 &#176;C.</li>
	<li>Using chilled pipette tips, pipette 80 &#181;L of basement membrane matrix into the center of each well of a 24-well culture plate to create a dome (Figure 1A). Place the plate in the incubator (37 &#176;C, 5% CO2) for 30 min to partially solidify the matrix.</li>
	<li>Prepare tissue: Using sterile dissection scissors, weigh 0.1 g of tissue from each sample and place it in PBS on ice.</li>
	<li>With sterile forceps, embed pre-weighed tissue into the partially solidified&#160;matrix dome. Add 30 &#181;L of basement membrane matrix on top of the embedded tissue. Return the plate to the incubator for 30 min to set the tissue in the basement membrane matrix. 
	NOTE: Embedding tissue in the basement membrane matrix is crucial to ensure that tissue remains in culture rather than floating. This preserves the health and quality of tissue (Figure 1B).</li>
	<li>Add 1 mL of growth media (DMEM with 10% FBS) to each well and return to the incubator.</li>
</ol>
4. Collagenase treatment
<ol>
	<li>Calculate the amount of collagenase solution needed. See Table 1.</li>
	<li>Prepare collagenase solution with DNase. Add 0.00017 g of DNase per 1 mL of collagenase solution. Vortex to mix.</li>
	<li>Using a razor blade, mince the isolated adipose tissue until the tissue appears homogeneous (Figure 2A). Transfer minced tissue to a sterile 50 mL conical tube. 
	NOTE: Do not over-mince. Mincing is complete when tissue appears homogeneous and paste-like. Under-minced tissue appears chunky and heterogeneous, and over-minced tissue will result in the release of free lipids (Figure 2B).</li>
	<li>Add prepared collagenase and DNase solution to the minced adipose tissue using volumes calculated in step 4.1. Vortex at maximum speed for 7-10 s to thoroughly combine. Place 50 mL conical tubes horizontally in a shaker set to 220 rpm, 37 &#176;C.</li>
	<li>Incubate shaking for 10 min, then vortex for 5 s and check the samples. Fully digested samples will appear homogeneous with no visible clumps or tissue pieces. If samples are not fully digested, continue digestion in the shaker, checking every 2 min (Figure 2C). 
	NOTE: Do not over-digest. Excessive time in collagenase solution can result in the breakdown of adipocytes and the release of lipids.</li>
	<li>Use a serological pipette to pass the digested tissue through a sieve into a new 50 mL conical. Add a volume of FBS equal to the volume of tissue + collagenase solution through the sieve into a conical to neutralize collagenase. 
	NOTE: A small amount of tissue will remain in the sieve after filtering.</li>
</ol>
5. Washing the tissue
NOTE: Complete all the following steps in a sterile tissue culture hood.
<ol>
	<li>Prepare 1 x 15 mL tube with 10 mL of warm culture media for each condition.</li>
	<li>Spin the tubes from step 4.6 at 300 x g for 3 min at the temperature specified in Table 1. 
	NOTE: The temperature for centrifugation needs to be modified based on the sex of the mice from which the adipose tissue was isolated; these temperatures are provided in Table 1.</li>
	<li>Ensure that after spinning, the tissue separates into four layers (Figure 3A). 
	NOTE: The topmost layer is the free lipids, the second layer is the mature adipocyte layer, the third layer is the collagenase and FBS, and the fourth layer is the stromal vascular fraction (SVF) that pellets to the bottom20,21.</li>
	<li>Carefully discard the top layer of free lipids with a 200 &#181;L pipette.</li>
	<li>After removing the top layer, use a p1000 pipette with the bottom cut off to transfer the mature adipocytes to the prepared 15 mL tube with 10 mL of growth media (Figure 3B).
	<ol>
		<li>Optional: To isolate the primary adipocyte progenitor cells (APCs), spin the tubes again at 300 x g for 10 min. Then proceed to the adipocyte progenitor cell isolation by Liu et al.22, Cho et al.23, or similar.</li>
	</ol>
	</li>
	<li>Invert the 15 mL tubes with the mature adipocytes gently 3-5 times until the white layer has dispersed throughout the media.</li>
	<li>Let the tubes rest upright for 10 min at the temperature indicated in Table 1 to allow for separation of the adipocyte and media layers.</li>
	<li>After the 10 min rest, spin the tubes at 100 x g for 1 min at room temperature to further promote separation of layers. Following the spin, the samples will be separated again into four layers, as shown in Figure 3A.</li>
	<li>Remove and discard the top layer of free lipids with a pipette.</li>
	<li>Using a 21 G needle attached to a 5 mL syringe, slide the needle down the edge of the tube until&#160;the bottom of the needle is clear of the mature adipocyte layer (Figure 3C). Ensure that the top of the needle and bottom of the syringe do not disturb the mature adipocyte layer.</li>
	<li>Slowly pull up the syringe to remove the culture media and any cellular debris. 
	NOTE: As the media is removed, ensure that the needle tip is clear of the mature adipocyte layer.</li>
	<li>Gently add 10 mL of culture media to each tube.</li>
	<li>Repeat steps 5.6-5.11. Do not add more culture media.</li>
	<li>Using gel-loading pipette tips, slide the pipette tip down the edge of the tube to remove the remaining media from below the mature adipocyte layer.</li>
</ol>
6. Packing
<ol>
	<li>Centrifuge cells at 100 x g for 1 min at the temperature indicated in Table 1. Remove free lipids (clear oil layer on top of cells) using a pipette. Remove the culture media below adipocytes using a gel loading tip. Take care to minimize disruption of the adipocyte layer (Figure 4).</li>
	<li>Repeat step 6.1 until all culture media and free lipids have been removed (typically 3-5 times). The cell pack should be white in color with no visible free lipids above cells or media below cells (Figure 4).</li>
</ol>
7. Plating and functional assays
<ol>
	<li>Plating the mature adipocytes for cell culture
	<ol>
		<li>Remove the cell culture inserts from a tissue culture-treated 24-well plate and place them with the membrane side up on the surface of the hood.</li>
		<li>Fill alternating wells of the plate with 1 mL warm culture media.</li>
		<li>Using a cut p200 pipette tip, place 30 &#181;L of packed mature adipocytes from step 6.2 onto the top of a transwell insert (Figure 5A,B).</li>
		<li>Carefully pick up and invert the transwell insert and place it into a prefilled well in the 24-well plate (Figure 5A). Carefully place the plate into the incubator set at 37 &#176;C with 5% CO2. 
		NOTE: Properly packed adipocytes should form an opaque layer with no visible clear free lipid droplets when viewed from above (Figure 5C).</li>
		<li>Change the media on the mature adipocytes every 2-7 days for up to 14 days.
		<ol>
			<li>To remove the media from the wells, hold the transwell in place, and use a p200 pipette to carefully remove the media through the small openings on the side of the transwell insert (Figure 5D).</li>
			<li>Once media has been removed, while still holding the transwell in place, very slowly add 1 mL of media to the side wall of the well. 
			NOTE: Take care to add and remove media very slowly. Adipocytes can easily become dislodged from the membrane if the media is changed quickly.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Lipolysis assay
	<ol>
		<li>Plate adipose tissue explants as described in step 3.4 or mature adipocytes as described in step 7.1. 
		NOTE: Cells or tissue must be cultured in phenol red-free media for at least 24 h before conducting lipolysis assay.</li>
		<li>Solution preparation: Prepare a solution of 5% BSA in DMEM (no phenol red) by dissolving 5 g of BSA in 100 mL of DMEM. Invert gently to combine until BSA is fully dissolved.
		<ol>
			<li>Prepare working solutions for control and stimulation media. For control media, combine 5% BSA DMEM media with the vehicle. For stimulation media, combine 5% BSA DMEM media with 5 &#181;M of CL-316,243. Warm the control and stimulation media to 37 &#176;C prior to use.</li>
		</ol>
		</li>
		<li>Remove media from wells and wash with 1 mL warm DPBS. Fill wells with 500 &#181;L per well of control or stimulation media. Incubate at 37 &#176;C, 5% CO2.</li>
		<li>At 1 h, 2 h, 4 h, and 24 h after initiating the lipolysis assay, collect 100 &#181;L of media from each well into a microcentrifuge tube and replace it with 100 &#181;L of the appropriate control or stimulation media. 
		NOTE: Samples can be stored at -20 &#176;C at this point.</li>
		<li>Run Glycerol Colorimetric Assay using a commercially available free glycerol kit (see Table of Materials) or similar.
		<ol>
			<li>Prepare reagents as directed in the kit.</li>
			<li>Thaw samples and mix gently by pipetting.</li>
			<li>Prepare a standard curve with a seven-point, 2-fold serial dilution using the glycerol standard solution.</li>
			<li>Transfer 25 &#181;L of sample or standard to each well of a clear-bottom 96-well plate.</li>
			<li>Add 100 &#181;L of free glycerol assay reagent to each well containing sample or standard solution.</li>
			<li>Incubate for 15 min at room temperature, then read the absorbance at 540 nm (Figure 6A-D).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Imaging
	<ol>
		<li>Prepare the imaging slides. To prepare slides, remove the backing paper layer from each imaging spacer and place it onto the slide. Two imaging spacers can be placed side-by-side on each slide.</li>
		<li>Using a cut 200 &#181;L pipette tip, transfer 30 &#181;L of packed adipocytes from step 6.2 into a microcentrifuge tube.</li>
		<li>Prepare the staining mix. Prepare 200 &#181;L per sample of plasma membrane staining solution at 5 &#181;g/mL and Hoechst at 4.5 &#181;g/mL in PBS. Add stain mix to samples and gently shake.</li>
		<li>Stain samples for 30 min at room temperature. Protect the samples from light.</li>
		<li>After the staining is complete, wash the samples. Add 500 &#181;L PBS to each sample and shake gently to combine. Let stand for 5 min. Spin at 100 x g for 1 min, then remove media from under the cells with a gel loading pipette tip.</li>
		<li>Prepare the slides. Remove the clear plastic film from the top side of the imaging spacers to expose the sticky layer.</li>
		<li>Add 500 &#181;L of PBS to the tube of stained adipocytes and mix gently.</li>
		<li>Quickly, so as to not allow time for the mature adipocytes to rise to the surface, use a cut p200 pipette tip to place 35 &#181;L of the adipocytes within the spacer on the imaging slide.</li>
		<li>Place a cover slip on the spacer.</li>
		<li>Immediately proceed to imaging (Figure 6C). A delay in imaging may result in bleeding of the membrane marker.</li>
	</ol>
	</li>
</ol>

Culturing Murine Adipocytes and Explants for Functional and Imaging Studies

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The authors have nothing to disclose.

Primary adipocytes are notoriously difficult to isolate and culture because they rupture easily, and their large size precludes the use of common primary cell isolation tools such as flow cytometry. The protocol described here yields clean and functional mature adipocytes, which can be used for a variety of downstream applications. Importantly, this protocol allows for the separation of the mature adipocytes from other cellular debris and the free lipid layer, allowing effective imaging, culturing, and lipolysis assays. The protocol can readily be combined with published protocols22,23 for the isolation of the stromal vascular fraction and APCs. This protocol also details a procedure for the preparation and culturing of whole-tissue explants from adipose tissue, which can be used for downstream applications such as lipolysis assays and the collection of secreted factors from whole adipose tissue.
Critical aspects include proper mincing, digestion, and removal of the free lipid layer. To ensure proper mincing, it is important to closely monitor the tissue&#39;s visual appearance throughout the process. The time and effort needed to achieve an optimal mince of the tissue will vary depending on the adipose tissue depot, dietary interventions (such as a high-fat diet), and the researcher. Other tools for mincing, such as scissors, can be incorporated if necessary, but this has not been explored here. Of note, over-mincing should be avoided as it will rupture adipocytes, which not only decreases the immediate yield but also decreases total yield and adipocyte quality due to the deleterious effect the free lipid layer has on the remaining adipocytes. Proper digestion, similar to a proper mince, will depend on the adipose tissue depot, dietary interventions, age of mice, etc. The addition of DNase to the collagenase helps to prevent the aggregation of the adipocytes during digestion, which can result from cellular debris. The digestion time for tissues is, therefore, highly variable. The provided estimates for digestion times serve as a guide, which can be found in Table 1. However, the best way to ensure the tissues are properly digested is to visually inspect the tissue throughout the process and as described in the protocol. The different centrifugation and wash temperatures detailed in Table 1, suggested based on sex, are essential for the proper separation of the mature adipocytes from both the free lipid layer as well as the other cellular debris and SVF; use of alternate temperatures may result in the loss of the mature adipocytes due to aggregation and adipocyte bursting.
The removal of the free lipid layer at every step is of critical importance, and failure to do so will present downstream complications for plating, imaging, and the lipolysis assay. Further, the sustained presence of the free lipid layer throughout the procedure will result in additional adipocyte death due to lipotoxicity, dramatically impacting overall yield and quality. While maximizing the efficiency of the mature adipocyte isolation is generally desired, removing the free lipid layer, even at the expense of some of the mature adipocytes, will result in an overall increase in mature adipocyte yield at the end of the protocol.
Limitations of the procedure include the number of samples. Due to the rigor and time consumed by the removal of media and free lipid layer, the procedure is generally limited to six samples at a time. Processing significantly more samples will result in primary adipocytes being exposed to free lipids for too long and decrease the overall yield. However, additional personnel may allow for an increase in the number of samples. The time from mincing to functional assay is critical as excessive delays in the removal of media and free lipid layer result in additional mature adipocyte bursting. The protocol is further limited in the ability to separate sub-populations of mature adipocytes.
Current methods to study mature adipocytes primarily rely on the isolation of APCs and the subsequent ex vivo differentiation of these cells. Additional methods, including organ-on-a-chip model and trans well co-cultures, have begun to be developed18,24. However, these ex vivo differentiated adipocytes have been shown to be transcriptionally and morphologically distinct from the mature adipocytes in the adipose tissue17. Morphologically, ex vivo differentiated adipocytes are multilocular, while in vivo isolated adipocytes are unilocular. This protocol was modified and optimized from previous reports17,25 to successfully isolate and culture mature adipocytes from as little as 0.5 g of adipose tissue compared to other protocols that require at least 5 g of adipose tissue25 . Finally, this study aimed to visually illustrate the successful completion of key steps in the protocol in lieu of concrete times, centrifugation temperatures, and collagenase amounts, though guidelines are included in Table 1. These parameters need to be adjusted according to factors such as the adipose tissue depot, the sex, and the adiposity of the animal, all of which can impact the efficiency of the digestion and clean separation of mature adipocytes in this protocol.
In total, the procedure takes 3-4 h, depending on the number of mice pooled and the number of individual adipose tissue samples. It is anticipated that the ability to culture and assess the functional role of mature adipocytes will enable many downstream applications for the study of adipocyte biology itself (e.g., metabolism, nutrient utilization, and tracing) and the interaction of adipocytes with other cell types, such as APCs, cancer cells, and cells of the immune system.

It is expected that the prevalence of adult obesity in the United States will reach 50% by 20301, making it imperative to better understand the cellular and physiological effects of obesity. Obesity is characterized by caloric imbalance resulting in the expansion of white adipose tissue due to increased storage of energy as fat. Adipose tissue expansion can occur both through the generation of new adipocytes (hyperplasia) or through an increase in the size of existing adipocytes (hypertrophy). Although both processes result in the expansion of adipose tissue, hypertrophic expansion coincides with reduced metabolic health and higher incidences of metabolic disorders2,3. A better understanding of the complexities of adipose tissue will provide insight into the molecular mechanisms regulating metabolic disorders and other obesity-related comorbidities, including type 2 diabetes and cancers.
Adipose tissue has long been appreciated for its role in energy storage and thermoregulation. More recently, however, adipose tissue has emerged as an important metabolic signaling tissue, responsible for the secretion of metabolites, lipids, and peptides4,5. These adipose tissue-derived molecules, termed adipokines, signal locally and systemically and can regulate energy homeostasis, immune function, vascular dynamics, and more5,6,7. Mature adipocytes make up approximately 50% of cells in adipose tissue and represent the large majority of adipose tissue volume8. Importantly, studies have found that adipocytes consist of many subpopulations9,10. As such, adipocyte progenitor cells generate distinct populations of mature adipocytes based on factors such as sex and adipose tissue depot. These mature adipocytes can be further modulated by changes in diet and obesity, creating subpopulations of adipocytes that are transcriptionally distinct and express different paracrine signatures9,11,12,13,14,15,16. Despite their complexity and physiologic importance, the ability to isolate and culture mature adipocytes remains a distinct barrier to studying these cells.
The currently available methods used to isolate mature adipocytes employ differentiation from adipocyte progenitor cells (APCs), fluorescent activated cell sorting, and centrifugation-based techniques, though all of these have their own drawbacks. In vitro or ex vivo differentiation of APCs into mature adipocytes is widely used to develop mature adipocytes. However, these artificially differentiated cells are multilocular, containing many small lipid droplets, rather than the unilocular phenotype of mature adipocytes in vivo, which contain one large lipid droplet17. Furthermore, studies have found that in vitro differentiated cells are transcriptionally distinct from isolated mature adipocytes found in vivo17. Importantly, in vitro differentiation is unable to capture the difference between lean and obese adipocytes. Fluorescent-activated cell sorting (FACS) is commonly used to isolate specific cell populations from tissue; however, mature adipocytes can easily burst under FACS conditions due to their large size and fragile membranes, making this an inefficient method for mature adipocyte isolation. Other existing protocols result in the accumulation of large quantities of free lipids, which can be toxic to mature adipocytes in culture over time.
Here, we describe a protocol for the isolation of murine mature adipocytes from white adipose tissue. This protocol was adapted from the protocol for mature adipocyte aggregate cultures originally established by Harms et al.17; however, the centrifugation-based approach described here uses common laboratory equipment and is capable of efficiently isolating adipocytes from small amounts (~0.5 g) of adipose tissue. This is an easily adaptable system, and modifications for isolating mature murine adipocytes from male and female mice, visceral and subcutaneous fat depots, and different diet conditions are included.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x Poloxamer 188 solution</td>
			<td>Sigma</td>
			<td>P5556-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical</td>
			<td>Cellstar</td>
			<td>188261</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G Needle</td>
			<td>BD</td>
			<td>305165</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conical</td>
			<td>Cellstar</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>6.5 mm Transwell with 0.4 &#181;m Pore Polycarbonate Membrane Insert</td>
			<td>Corning</td>
			<td>3413</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C1016-500gm</td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask Plasma Membrane Stains</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10045</td>
			<td></td>
		</tr>
		<tr>
			<td>CL 316243 disodium salt</td>
			<td>Tocris</td>
			<td>1499</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Powder</td>
			<td>Sigma</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM no pheonol Red</td>
			<td>Gibco</td>
			<td>A14430-01</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma-Aldrich</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>A56708-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Tissue Path Superfrost Plus Gold Slides</td>
			<td>Fisher scientific</td>
			<td>1518848</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Pipet Tip</td>
			<td>Fisher scientific</td>
			<td>02-707-181</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol Assay Kit</td>
			<td>Abcam</td>
			<td>AB133130</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks&#39; Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Sigma</td>
			<td>83264-100ml-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Megafuge 16R Centrifuge</td>
			<td>Thermo Scientific&#160;</td>
			<td>75004271</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342 Solution</td>
			<td>Thermo Fisher Scientific</td>
			<td>62249</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubating Mini Shaker</td>
			<td>VWR</td>
			<td>12620-942</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M4530</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses</td>
			<td>VWR</td>
			<td>16004-302</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>SecureSeal Imaging Spacers</td>
			<td>Grace Biolabs</td>
			<td>654006</td>
			<td></td>
		</tr>
		<tr>
			<td>Seive (SteeL Fine Mesh Cocktail Strainer)</td>
			<td>OXO</td>
			<td>3112000</td>
			<td>https://www.oxo.com/steel-fine-mesh-cocktail-strainer-660.html</td>
		</tr>
	</tbody>
</table>

isolation and culturing of primary murine adipocytes from lean and obese mice

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and mobilization, thermoregulation, and the secretion of endocrine factors. The expansion of white adipose tissue due to caloric imbalance results in both the enlargement of existing adipocytes and the generation of additional adipocytes from adipocyte progenitor cells. Obesity-driven changes to white adipose tissue, including those affecting adipocytes, are associated with numerous comorbidities, such as type 2 diabetes and 13 types of cancer. A significant barrier to studying how adipocytes contribute to disease is the inability to readily isolate and culture mature adipocytes. This article describes a protocol to isolate murine lean and obese adipocytes from the subcutaneous and visceral fat depots of male and female C57BL/6 mice. The protocol details how isolated primary adipocytes can be cultured in a membrane adipocyte aggregate system for up to 2 weeks, facilitating their functional analysis in co-culture experiments, lipolysis assays, or through the collection of conditioned media containing adipocyte-secreted factors. Additionally, the protocol outlines methods for culturing adipose tissue explants in basement membrane matrix domes and imaging primary isolated adipocytes. Importantly, this approach can be integrated with existing protocols for the isolation of adipose tissue-resident adipocyte&#160;progenitor cells using fluorescence-activated cell sorting (FACS). Together, these protocols provide researchers with tools to functionally study adipocytes, adipocyte progenitor cells, and whole adipose tissue from lean and obese, male and female mice.

As shown in Figure 2, proper mincing (Figure 2B) and digestion (Figure 2C) are important for the isolation of intact mature adipocytes. Under-mincing and under-digestion can result in a low yield of mature adipocytes due to inadequate separation from the connective tissue. Visually, this can be observed with stringy tissue remaining after mincing and visible clumps in collagenase solution. Additionally, over-mincing and over-digesting also result in low yield due to the bursting of the mature adipocytes. Visually, this results in the loss of the white opaque layer and an accumulation of a large, clear, free lipid layer. Inappropriate levels of mincing or digestion will result in poor adipocyte isolation.
Proper preparation and digestion of adipose tissue will result in the separation of the tissue into four distinct layers, as seen in Figure 3. Some bursting of the adipocytes resulting in the release of free lipids is inevitable;&#160;however, proper mincing and digestion will ensure minimal adipocyte rupture. The remaining free lipid layer and media will be removed during the packing of the adipocytes (Figure 4). The proper packing of the adipocytes is essential for the efficient isolation of mature adipocytes. Any remaining free lipids or media will result in poor isolation and negatively impact downstream functional assays, such as plating, lipolysis, and cell imaging.
This protocol generates a clean preparation of mature adipocytes. Cell imaging and lipolysis assays can be performed to assess cellular function and demonstrate that the mature adipocytes retain&#160;function post-isolation and after culturing for up to two weeks. The lipolysis assay indicates that the mature adipocytes maintain the ability to respond to cellular signaling through the release of free glycerol upon stimulation of the &#946;-3 adrenergic receptor (Figure 6A,B). This ability is retained even after 2 weeks in culture and is highly comparable to the lipolysis activity of adipose tissue explants cultured for 24 h (Figure 6C,D). Poor-quality adipocytes will show attenuated lipolysis. The cell imaging of isolated adipocytes shows round and intact unilocular mature adipocytes containing a single large lipid droplet. Poor-quality adipocyte isolations will show primarily large, extracellular oil droplets indicative of burst adipocytes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67846/67846fig01.jpg" /> 
Figure 1: Embedding of adipose tissue explants. (A) Image of partially-solidified basement membrane matrix dome prior to embedding the tissue. (B) Representative images of improper and proper tissue embedding. Improper embedding is visible as the tissue appears to float partially or completely above the dome. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67846/67846fig01large.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/67846/67846fig02.jpg" /> 
Figure 2: Schema of proper mincing and digestion of adipose tissue. (A) Schematic representation of tissue mincing and digestion steps 4.2-4.6. (B) Representative images of under, optimal, and over-minced tissue. Under-minced tissue has large chunks that retain their original shape. Over-minced tissue results in a visible increase in the free lipids surrounding the tissue, which appear clear and oily. (C) Representative images of under, optimal, and over-digested tissue. Under-digested tissue contains chunks of whole tissue remaining in the solution. Over-digested tissue exhibits a large free lipid layer that quickly separates from the solution. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67846/67846fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67846/67846fig03.jpg" /> 
Figure 3: Separation of layers of digested adipose tissue. (A) Schematic and representative image of the layers that are present following step 5.3. (B) Use of a cut pipette tip to remove the mature adipocyte layer from the 50 mL conical tube. (C) Proper technique and placement for the needle and syringe to remove the culture media from below the mature adipocyte layer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67846/67846fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67846/67846fig04.jpg" /> 
Figure 4: Sequential packing of mature adipocytes. Sequential images of packing the mature adipocytes. Layers of free lipids, adipocytes, and media are labeled. Following each spin, free lipids and media are removed. This is repeated until no media or free lipids separate from the adipocytes after a spin. The final image is representative of well-packed mature adipocytes. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67846/67846fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67846/67846fig05.jpg" /> 
Figure 5: Plating and culturing of mature adipocytes. (A) Schematic of the plating of mature adipocytes. Using a cut p200 pipette tip, packed adipocytes are placed onto the membrane of a cell culture insert. The insert is then inverted into pre-filled wells of culture media. (B) A representative image of plating mature adipocytes depicting cut pipette tip depositing adipocytes on transwell insert. (C) Schematics and representative images of properly packed and poorly packed adipocytes plated with transwells, as seen from above the wells. Properly packed adipocytes appear white and opaque, while poorly packed adipocytes result in the formation of clear lipid droplets. (D) Schematic of the technique used to change culture media. A p200 pipette tip is used to carefully remove the media through the openings in the side wall of the transwell insert. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67846/67846fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67846/67846fig06.jpg" /> 
Figure 6: Imaging of mature adipocytes and lipolysis assay. (A,B) Glycerol release from subcutaneous (A) and visceral (B) mature adipocytes. Adipocytes were isolated from 16-week-old C57BL/6 male mice fed a normal chow diet. 30 &#181;L of adipocytes were plated as described in step 7.1 and cultured for 1 or 10 days. Adipocytes were treated with 5 &#181;M of CL-316,243 for 1 h, 2 h, and 4 h, and supernatants were collected and analyzed for free glycerol. (C,D) Glycerol release from subcutaneous (C) and visceral (D) adipose tissue explants. Adipose tissue was isolated from 18-week-old C57BL/6 female mice fed a normal chow diet. 0.1 g of adipose tissue explants were plated as described in steps 3.1-3.4 and cultured for 24 h. Adipocytes were treated with 5 &#181;M of CL-316,243 for 1 h, 2 h, and 4 h, and supernatants were collected and analyzed for free glycerol. N = 3 technical replicates for A-D. All data are mean &#177; standard deviation. (E) Representative images of isolated subcutaneous and visceral mature adipocytes. Adipocytes were isolated from 16-week-old C57BL/6J male mice fed a normal chow diet. Adipocytes were stained with plasma membrane stain (red) and Hoechst nuclei stain (blue). Scale bars: 50 &#181;m; magnification: 60x. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67846/67846fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sex</td>
			<td>Diet</td>
			<td>Depot</td>
			<td>Mincing difficulty</td>
			<td>Collagenase: Tissue ratio</td>
			<td>Suggested digestion time (min)</td>
			<td>Spin temp.</td>
			<td>Wash temp.</td>
		</tr>
		<tr>
			<td rowspan="4">Female</td>
			<td rowspan="2">Normal chow</td>
			<td>Subcutaneous</td>
			<td>Hard</td>
			<td>2:1</td>
			<td>12</td>
			<td>RT</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td>Visceral</td>
			<td>Medium</td>
			<td>1:1</td>
			<td>8</td>
			<td>RT</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td rowspan="2">High fat diet</td>
			<td>Subcutaneous</td>
			<td>Easy</td>
			<td>1:1</td>
			<td>8</td>
			<td>RT</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td>Visceral</td>
			<td>Easy</td>
			<td>1:1</td>
			<td>8</td>
			<td>RT</td>
			<td>37 &#176;C</td>
		</tr>
		<tr>
			<td rowspan="4">Male</td>
			<td rowspan="2">Normal chow</td>
			<td>Subcutaneous</td>
			<td>Hard</td>
			<td>2:1</td>
			<td>14</td>
			<td>4 &#176;C</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Visceral</td>
			<td>Medium</td>
			<td>1:1</td>
			<td>10</td>
			<td>4 &#176;C</td>
			<td>RT</td>
		</tr>
		<tr>
			<td rowspan="2">High fat diet</td>
			<td>Subcutaneous</td>
			<td>Easy</td>
			<td>1:1</td>
			<td>10</td>
			<td>4 &#176;C</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Visceral</td>
			<td>Easy</td>
			<td>1:1</td>
			<td>10</td>
			<td>4 &#176;C</td>
			<td>RT</td>
		</tr>
	</tbody>
</table>
Table 1: Sex, depot, and diet-specific modifications. The mincing difficulty of each tissue, described as &#34;Easy&#34;, &#34;Medium&#34;, or &#34;Hard&#34; in relation to other depots. Note that actual mincing time will vary depending on the researcher and tissue. Collagenase: Tissue ratio listed as volume (mL) of collagenase + DNase solution prepared in step 4.1 to the weight of tissue (g). Digestion time is an approximate estimate, but actual digestion time will vary according to mincing and tissue. Researchers should assess digestion as described in the protocol. Spin temperature and wash temperature differ between male and female mice. RT denotes room temperature.

Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Adipose tissue is primarily composed of mature, lipid-laden adipocytes by volume. These postmitotic cells play a critical role in energy storage and ...

isolation-culturing-primary-murine-adipocytes-from-lean-obese

Nanyang Technological University

Research

JoVE Journal

Biology

503 Views.  University of Utah School of Medicine. The size and fragility of mature adipocytes have limited the techniques and tools available for their study and isolation. Here, a protocol is described for the isolation of mature murine adipocytes, which can be easily adapted for different adipose depots and mouse diets.

Video: Isolation and Culturing of Primary Murine Adipocytes from Lean and Obese Mice

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

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 ...

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 of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

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 ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...

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

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

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

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

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

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.

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 and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...