This study was supported from the Instituto Nacional de Perinatologia (grant numbers: 3300-11402-01-575-17 and 212250-3210-21002-06-15) and CONACyT, Fondo Sectorial de Investigacion en Salud y Seguridad Social (FOSISS) (grant number 2015-3-2-61661).

This protocol was approved by the IRB of the Instituto Nacional de Perinatologia (212250-3210-21002-06-15). Participation was voluntary, and all the enrolled women signed the informed consent form.
1. Visceral adipose tissue collection
<ol>
	<li>Obtain VAT biopsies through partial omentectomy during cesarean section from healthy adult women with singleton pregnancies at term without labor.</li>
	<li>After the uterine closure and hemostasis, proceed to identify greater omentum and extend it on a wet compress. The AT exposed is VAT.</li>
	<li>Locate the largest blood vessel and trace an imaginary line of 7 x 5 cm to the greater omentum base.</li>
	<li>Identify an avascular zone and use Kelly forceps to drill the outer side of VAT.</li>
	<li>Use Ochsner forceps to clamp the proximal and distal side in the zone where VAT was drilled and use Metzenbaum scissors to cut the tissue.</li>
	<li>Tie greater omentum with black silk number 1.</li>
	<li>Remove Ochsner forceps and evaluate the hemostasis.</li>
	<li>Place the VAT biopsy into a sterile container and transport it to the lab immediately.</li>
</ol>
2. Enzymatic digestion of visceral adipose tissue and isolation of mature adipocytes and stromal vascular fraction cells
<ol>
	<li>Weigh the VAT biopsy and rinse with 1x PBS pH 7.4.</li>
	<li>Cut 4 g of VAT and use scissors to mince the tissue into small pieces in a dissection tray.</li>
	<li>Transfer minced VAT to a sterile 50 mL centrifuge tube, add 20 mL of PBS, and shake gently to remove the excess of red blood cells.</li>
	<li>Discard PBS and repeat the procedure twice.</li>
	<li>Transfer VAT to a new sterile 50 mL centrifuge tube and add 25 mL of digestion solution (0.25% collagenase type II, 5 mM glucose, 1.5% albumin in PBS).</li>
	<li>Incubate at 37 &#176;C for 60 min in an orbital shaker at 125 rpm.</li>
	<li>Filter the digested tissue through three layers of gauze into a new sterile 50 mL centrifuge tube.</li>
	<li>Centrifuge at 200 x g for 5 min at 4 &#176;C.</li>
	<li>Two phases are obtained, an upper phase that corresponds to mature adipocytes, while SFV cells remain in the pellet. Gently transfer the mature adipocytes into a new sterile 50 mL centrifuge tube using a transfer pipette.</li>
	<li>Add 20 mL of cold PBS, shake gently, and centrifuge at 200 x g for 5 min at 4 &#176;C.</li>
	<li>Discard PBS and repeat the procedure twice.</li>
	<li>Use a transfer pipette to gently transfer the mature adipocytes into a 1.5 mL DNase-RNase free microcentrifuge tube.</li>
	<li>Centrifuge at 200 x g for 1 min at 4 &#176;C and remove the excess PBS using a P200 micropipette. Repeat this step if it is necessary.</li>
	<li>Gently transfer 300 &#956;L of mature adipocyte suspension into a 1.5 mL DNase-RNase free microcentrifuge tubes. Store mature adipocytes at -80 &#176;C until RNA extraction. 
	NOTE: Adipocytes do not form a pellet.</li>
	<li>Once adipocytes are separated (step 2.9), aspirate most of the digestion solution with a transfer pipette, keeping the SVF pellet at the bottom of the tube.</li>
	<li>Transfer the pellet with SVF cells into a new 50 mL centrifuge tube using a transfer pipette.</li>
	<li>Wash cell pellet gently, resuspending in 20 mL of cold 1x PBS by pipetting up and down.</li>
	<li>Centrifuge at 800 x g for 5 min at 4 &#176;C and rapidly discard the supernatant by decantation.</li>
	<li>Perform a second wash by repeating steps 2.17 and 2.18.</li>
	<li>Add 5 mL of red blood cell lysis buffer equilibrated to room temperature (RT) to the SVF pellet and suspend by repeated pipetting. Do not vortex.</li>
	<li>Incubate for 5 min at RT and centrifuge for 5 min at 800 x g. Carefully remove the supernatant using a transfer pipette and dispose of properly.</li>
	<li>To neutralize the lysis buffer, add 10 mL of cold 1x PBS and gently mix until the cell pellet disaggregates.</li>
	<li>Centrifuge at 800 x g for 5 min to obtain SVF pellet and discard PBS. A white pellet should be visible at the bottom of the tube.</li>
	<li>Resuspend cells in 5 mL of 1x PBS at RT by repeated pipetting and filtered through three layers of gauze into a new 50 mL conical tube. Subsequently, these cells will be used for macrophage subpopulation characterization by flow cytometry.</li>
</ol>
3. RNA extraction from mature adipocytes
<ol>
	<li>Prepare a clean area, using RNase decontamination spray to avoid RNA degradation. Use a set of pipettes reserved for RNA procedures. All tubes and tips employed should be RNase-free.</li>
	<li>Thaw the mature adipocyte suspension (section 2.14) at 4 &#176;C if it was stored to -80 &#176;C. 
	NOTE: Avoid multiple freeze-thaw cycles.</li>
	<li>Lyse cells by adding 1,000 &#956;L of acid-guanidinium-phenol based reagent, and mix thoroughly with a P1000 micropipette to homogenize.</li>
	<li>Incubate for 5 min at RT to boost dissociation of nucleoprotein complexes.</li>
	<li>Centrifuge cell lysate for 5 min at 12,000 x g at RT. Three phases will be obtained: an upper phase (yellow) corresponding to adipocyte lipids, a middle phase (pink) corresponding to nucleic acids, and a pellet (cellular debris).</li>
	<li>Carefully remove the lipid layer, using a P200 micropipette.</li>
	<li>Transfer the middle phase into a new 2 mL tube, avoiding lipid remnant and pellet disturbing (approximately 700 &#956;L).</li>
</ol>
4. Purification of total RNA, including microRNAs
NOTE: A column-based total RNA purification method is used to obtain high-quality total RNA.
<ol>
	<li>Add an equal volume of ethanol (95%&#8211;100%) to the sample from section 3.7, approximately 700 &#956;L, and mix by hand inverting the tube for 10 s.</li>
	<li>Transfer 700 &#956;L of the mixture into a column inserted in a collection tube, centrifuge and discard the flow-through.</li>
	<li>Reload the column and repeat step 4.2.</li>
	<li>Transfer the column into a new collection tube.</li>
	<li>Add 400 &#181;L of pre-wash buffer to the column and centrifuge. Discard the flow-through and repeat this step.</li>
	<li>Add 700 &#181;L of wash buffer to the column and centrifuge for 2 min.</li>
	<li>Transfer the column carefully into a nuclease-free tube.</li>
	<li>Add 50 &#181;L of nuclease-free water directly to the column matrix and elute the RNA by centrifugation. Prepare aliquots of 10 &#181;L using 200 &#181;L microfuge tubes.</li>
	<li>Immediately put aliquots on ice and use one of them to determine RNA concentration and purity.</li>
	<li>Prepare an aliquot of 5 &#181;L of total RNA adjusted to 100 ng/&#181;L to measure RNA integrity and microRNA concentration.</li>
	<li>Store at -80 &#176;C until use.</li>
</ol>
5. Determination of mature adipocyte RNA concentration, purity, and integrity; microRNA quantification assay
NOTE: RNA concentration and purity determinations are performed using a UV-Vis spectrophotometer; RNA integrity and miRNA quantification are performed using an RNA quality control analyzer.
<ol>
	<li>Wash the sample reader with molecular grade water and wipe.</li>
	<li>Load 2 &#181;L of elution water (blank), change the setting to RNA, and click on the Blank button.</li>
	<li>Load 2 &#181;L of sample and click on the Measure button.</li>
	<li>After the reading is complete, record the A260/A280 and A260/A230 ratios as well as the amount of RNA (ng/&#181;L) (Figure 1A). 
	NOTE: RNA with OD260/OD280 and OD260/OD230 ratio around 2.0 is considered pure.</li>
	<li>Measure the RNA integrity number (RIN) using an RNA integrity kit following the manufacturer&#180;s instructions (Figure 1B). 
	NOTE: A RIN =10 corresponds to intact RNA, whereas a RIN &#8804; 3.0 indicates a strongly degraded RNA. For expression microarrays or RT-qPCR, use RNA with RIN &#8805; 6.0.</li>
	<li>Use a small RNA kit to determine the concentration and percent of microRNAs, following the manufacturer&#180;s instructions (Figure 1C). 
	NOTE: To avoid the overestimation of small and micro RNAs, use RNA samples with RIN &#8805; 6.0.</li>
</ol>
6. Count and viability of stromal vascular fraction cells
<ol>
	<li>Dilute 10 &#956;L of SVF cell suspension (section 2.24) into 90 &#956;L of 0.4% Trypan Blue Solution (final dilution 1:10) and apply 10 &#956;L to a standard hemocytometer.</li>
	<li>Count the viable cells carefully, excluding the dead cells, in four squares at the corner of the counting chamber.</li>
	<li>Determine the cell concentration present in the original suspension: Cell concentration = Total cell count/4 x dilution factor (10) x 10,000 = Cells/mL</li>
	<li>To calculate the cellular yield (cells per gram of tissue) obtained, multiply the cellular concentration by the original total sample volume (in mL) and divide by the weight of tissue digested (in grams).</li>
	<li>Evaluate cell viability as the percentage of the living cells as follows: % Viability = (Number of viable cells / Total number of cells) x 100.</li>
</ol>
7. Characterization of macrophage subsets from stromal vascular fraction
<ol>
	<li>Transfer a total of 1 x 106 SVF cells/mL to a 5 mL round-bottom polypropylene test tube for flow cytometry and pellet cells by centrifugation at 800 x g for 5 min at 4 &#176;C.</li>
	<li>Vortex carefully to loosen the pellet and resuspend the cells in 100 &#956;L of 1x PBS at RT.</li>
	<li>Add pre-titrated optimal concentration of each fluorochrome-conjugated monoclonal antibody specific for a cell surface antigen; mix gently and incubate for 15 min in the dark at RT.</li>
	<li>Add an excess of cold 1x PBS (&#8776; 1 mL) and centrifuge the SVF at 400 x g for 5 min at 4 &#176;C.</li>
	<li>Discard supernatants rapidly by decantation. Be careful not to disturb the pellet.</li>
	<li>Add 500 &#956;L of 1x lysing solution and incubate 15 min at RT, protecting the tubes from direct light.</li>
	<li>Remove the solution after centrifuge at 400 x g for 5 min at 4 &#176;C, and store at 2&#8211;8 &#176;C in the dark until data acquisition.</li>
	<li>Vortex the cells thoroughly at low speed to reduce aggregation before acquiring.</li>
	<li>Resuspend the SVF pellet in 5 mL of sheath fluid at RT, and subsequently filter through three layers of gauze into a new 5 mL round-bottom polypropylene test tube before analysis by flow cytometry, to reduce clogging the cell sorter lines.</li>
	<li>Vortex each tube briefly before analysis.</li>
	<li>Acquire data from the samples of interest. For flow analysis, count a minimum of 10,000 events. 
	NOTE: If using a cell sorting flow cytometer, separate the macrophages for application in subsequent studies.</li>
</ol>
8. Gating strategy
<ol>
	<li>Plot the height or width against the area for Forward Scatter (FSC) to determine the singlet population (Figure 2A).</li>
	<li>Select cells plotting the area for Side Scatter (SSC) against FSC, discarding the cellular debris (Figure 2B).</li>
	<li>From selected cells, identify the populations expressing CD45 as hematopoietic cells (Figure 2C), and CD45/CD14 double positive cells as macrophages (Figure 2D).</li>
	<li>From CD45+/CD14+ macrophages, detect negative and positive HLA-DR cells (Figure 2E). 
	NOTE: Include the compensation controls for the antibody panel and adjust spectral overlap on a multicolor flow cytometer adequate for the panel. We perform flow cytometry analysis using a cytometer equipped with three lasers (405 nm violet laser, 488 nm blue laser, and 640 nm red laser), and detectors for the indicated fluorochromes.</li>
</ol>

The authors have no conflicts of interest to disclose.

VAT plays a crucial role in metabolic regulation and inflammation. Increasing interest in the role of adipocytes and immune cells in the chronic inflammation associated with obesity has led to the development of different techniques to separate the SVF and fat cells present in AT. However, most techniques do not allow to obtain these two different sets of cells viable for downstream applications from the same VAT biopsy in a single procedure, which could be crucial for studies regarding interactions between adipocytes an...

White adipose tissue is composed not only of fat cells or adipocytes, but also of a non-fat cell fraction known as stromal vascular fraction (SVF), which contains a heterogeneous cell population consisting in macrophages, other immune cells as regulatory T cells (Tregs), and eosinophils, preadipocytes, and fibroblasts, surrounded by vascular and connective tissue1,2. Adipose tissue (AT) is now considered an organ that regulates physiological processes related to metabolism and inflammation through adipokines, cytokines, and microRNAs produced and released by different cells into the tissue, with autocrine, paracrine, and endocrine effects3,4. In humans, white adipose tissue comprises the subcutaneous adipose tissue (SAT) and the visceral adipose tissue (VAT), with important anatomic, molecular, cellular, and physiological differences between them2,5. SAT represents up to 80% of human AT, while VAT is located within the abdominal cavity, mainly in the mesentery and omentum, being metabolically more active6. Moreover, VAT is an endocrine organ that secretes mediators with a substantial impact on body weight, insulin sensitivity, lipid metabolism, and inflammation. Consequently, VAT accumulation leads to abdominal obesity and obesity-related diseases such as type-2 diabetes, metabolic syndrome, hypertension, and cardiovascular disease risk, representing a better predictor of obesity-associated mortality6,7,8,9.
In homeostatic conditions, adipocytes, macrophages, and the other immune cells cooperate to maintain the VAT metabolism through the secretion of anti-inflammatory mediators10. However, excessive VAT expansion promotes the recruitment of activated T cells, NK cells, and macrophages. In fact, in lean VAT, the ratio of the macrophages is 5%, while this ratio rises up to 50% in obesity, with macrophage polarization from anti-inflammatory to pro-inflammatory phenotype, generating a chronic inflammatory environment10,11.
As a consequence of the obesity pandemic, an astonishing number of reports have arisen addressing different VAT research topics, including adipocyte biology, epigenetics, inflammation, endocrine properties, and emerging areas as extracellular vesicles, among others8,10,12,13. However, although the VAT environment is defined by crosstalk between adipocytes and the resident or arriving macrophages, most studies have focused on only one cell population, and there is scarce information about the interaction of these cells in VAT and their pathophysiological consequences11,14. Moreover, valuable studies addressing the adipocyte-macrophage interplay in AT were performed using cell lines, lacking the in vivo priming conditions11,14,15. A suitable strategy to dissect the interaction or the particular contribution of these cells in VAT requires the isolation of both cell types from the same fat biopsy to perform in vitro assays that mirror as similar as possible the in vivo properties that regulate VAT metabolism.
Although the non-enzymatic dissociation methods based on mechanical forces to break the AT ensure minimal manipulation, these methods cannot be used if the aim is to study SVF cells, as they have lower efficiency in cell recovery and low cell viability compared to enzymatic methods, and a larger volume of tissue is needed16,17. Enzymatic digestion using collagenase is a gentle method that allows adequate digestion of collagen and extracellular matrix proteins of fibrous tissues such as WAT18 and is frequently used when trypsin is ineffective or damaging19. The protocol provides fundamental troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF cells from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique, giving information to ensure high yields (quantity, purity, and integrity) of total RNA from mature adipocytes, including microRNAs, for downstream expression applications. Simultaneously, the protocol is optimized to identify macrophage subsets from SVF cells through the staining of multiple membrane-bound markers for further analysis by flow cytometry20.

<table><tbody><tr><td>0.2 mL PCR tubes</td><td>Axygen</td><td>PCR-02-C</td><td>RNase, DNase free and nonpyrogenic</td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>Axygen</td><td>MCT-150-C</td><td>RNase, DNase free and nonpyrogenic</td></tr><tr><td>10 mL serological pipettes</td><td>Corning</td><td>CLS4101-50EA</td><td>Individually plastic wrapped</td></tr><tr><td>10 &amp;micro;L universal pipet tip</td><td>Axygen</td><td>T-300-L-R</td><td>RNase, DNase free and nonpyrogenic</td></tr><tr><td>10 &amp;micro;L universal pipet tip</td><td>Axygen</td><td>T-300-R-S</td><td>RNase, DNase free and nonpyrogenic</td></tr><tr><td>1000 &amp;micro;L universal pipet tip</td><td>Axygen</td><td>T-1000-B-R</td><td>RNase, DNase free and nonpyrogenic</td></tr><tr><td>2.0 mL microcentrifuge tube</td><td>Axygen</td><td>MCT-200-C</td><td>RNase, DNase free and nonpyrogenic</td></tr><tr><td>200 &amp;micro;L universal pipet tip</td><td>Axygen</td><td>T-200-Y-R</td><td>RNase, DNase free and nonpyrogenic</td></tr><tr><td>2100 Bioanalyzer Instrument</td><td>Agilent</td><td>G2939BA</td><td>-</td></tr><tr><td>2101 Bioanalyzer PC</td><td>Agilent</td><td>G2953CA</td><td>2100 Expert Software pre-installed in PC</td></tr><tr><td>5 ml Round Bottom Polystyrene Test Tube</td><td>Corning</td><td>352003&amp;nbsp;</td><td>Snap cap, sterile</td></tr><tr><td>50 mL centrifuge tubes</td><td>Corning</td><td>CLS430828-100EA</td><td>Polipropilene, conical bottom and sterile</td></tr><tr><td>Acid-guanidinium-phenol based reagent</td><td>Zymo Research</td><td>R2050-1-200</td><td>TRI Reagent or similar</td></tr><tr><td>Agilent RNA 6000 Nano Kit</td><td>Agilent</td><td>5067-1511</td><td>-</td></tr><tr><td>Agilent Small RNA Kit</td><td>Agilent</td><td>5067-1548</td><td>-</td></tr><tr><td>APC/Cy7 anti-human CD14 Antibody</td><td>BioLegend</td><td>325620</td><td>0.4 mg/10&lt;sup&gt;6&lt;/sup&gt; cells, present on monocytes/macrophages, clone HCD14</td></tr><tr><td>Baker</td><td>-</td><td>-</td><td>250 ml, non sterile</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A3912-100G</td><td>Heat shock fraction, pH 5.2, &amp;ge;96%</td></tr><tr><td>Chip priming station</td><td>Agilent</td><td>5065-9951</td><td>-</td></tr><tr><td>Collagenase type II</td><td>Gibco</td><td>17101-015</td><td>Powder</td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td>Powder</td></tr><tr><td>Direct-zol RNA Miniprep</td><td>Zymo Research</td><td>R2051</td><td>Supplied with 50 mL TRI reagen</td></tr><tr><td>Dissecting forceps</td><td>-</td><td>-</td><td>Steel, serrated jaws and round ends</td></tr><tr><td>Dissection tray</td><td>-</td><td>-</td><td>Stainless steel</td></tr><tr><td>Ethyl alcohol</td><td>Sigma-Aldrich</td><td>E7023-500ML</td><td>200&amp;nbsp;proof, for molecular biology</td></tr><tr><td>FACS Flow Sheath Fluid</td><td>BD Biosciences</td><td>342003</td><td>-</td></tr><tr><td>FACS Lysing Solution</td><td>BD Biosciences</td><td>349202</td><td>-</td></tr><tr><td>FACSAria III Flow Cytometer/Cell Sorter</td><td>BD Biosciences</td><td>648282</td><td>-</td></tr><tr><td>FASCDiva Software</td><td>BD Biosciences</td><td>642868</td><td>Software v6.0 pre-installed</td></tr><tr><td>Hemacytometer</td><td>Sigma</td><td>Z359629-1EA</td><td>-</td></tr><tr><td>Manual cell counter</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Mayo dissecting scisors</td><td>-</td><td>-</td><td>Stainless steel</td></tr><tr><td>Microcentrifuge</td><td>-</td><td>-</td><td>Adjustable temperature</td></tr><tr><td>Nanodrop spectrophotometer</td><td>Thermo Scientific</td><td>ND2000LAPTOP</td><td>-</td></tr><tr><td>Orbital shaker</td><td>-</td><td>-</td><td>Adjustable temperature and speed</td></tr><tr><td>P10&amp;nbsp;variable volume&amp;nbsp;micropipette&amp;nbsp;</td><td>Thermo Scientific-Finnpipette</td><td>4642040</td><td>1 to 10 &amp;mu;L</td></tr><tr><td>P1000&amp;nbsp;variable volume&amp;nbsp;micropipette&amp;nbsp;</td><td>Thermo Scientific-Finnpipette</td><td>4642090</td><td>100 to 1000 &amp;mu;L</td></tr><tr><td>P2&amp;nbsp;variable volume&amp;nbsp;micropipette&amp;nbsp;</td><td>Thermo Scientific-Finnpipette</td><td>4642010</td><td>0.2 to 2 &amp;mu;L</td></tr><tr><td>P200&amp;nbsp;variable volume&amp;nbsp;micropipette&amp;nbsp;</td><td>Thermo Scientific-Finnpipette</td><td>4642080</td><td>20 to 200 &amp;mu;L</td></tr><tr><td>PCR tube storage rack</td><td>Axygen</td><td>R96PCRFSP</td><td>-</td></tr><tr><td>PE/Cy5 anti-human HLA-DR Antibody</td><td>BioLegend</td><td>307608</td><td>0.0625 mg/10&lt;sup&gt;6&lt;/sup&gt; cells, present on macrophages, clone L243</td></tr><tr><td>PE/Cy7 anti-human CD45 Antibody</td><td>BioLegend</td><td>304016</td><td>0.1 mg/10&lt;sup&gt;6&lt;/sup&gt; cells, present on leukocytes, clone H130</td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P3813-10PAK</td><td>Powder, pH 7.4, for preparing 1 L solutions</td></tr><tr><td>Pipette controller</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Red Blood Cells Lysis Buffer</td><td>Roche</td><td>11 814 389 001</td><td>For preferential lysis of red blood cells from human whole blood</td></tr><tr><td>Refrigerated centrifuge</td><td>-</td><td>-</td><td>Whit adapter for 50 mL conical tubes</td></tr><tr><td>Sterile Specimen container</td><td>-</td><td>-</td><td>-</td></tr><tr><td>Transfer pipette</td><td>Thermo Scientific-Samco</td><td>204-1S</td><td>Sterile</td></tr><tr><td>Trypan Blue</td><td>Gibco</td><td>15250-061</td><td>0.4% Solution</td></tr><tr><td>Tube racks</td><td>-</td><td>-</td><td>For different tube sizes</td></tr><tr><td>Vortex Mini Shaker</td><td>Cientifica SENNA</td><td>BV101</td><td>-</td></tr></tbody></table>

isolation of viable adipocytes and stromal vascular fraction from human visceral adipose tissue suitable for rna analysis and macrophage phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol describes an enzymatic method using collagenase digestion followed by differential centrifugation to isolate, in a single process, viable mature adipocytes and SVF cells from VAT biopsies obtained from healthy pregnant women after partial omentectomy. In this case, we use the adipocytes for RNA extraction and the SVF for macrophage phenotyping.
The RNA extraction protocol enabled to obtain RNA with an adequate purity high integrity, and microRNAs from mature adipocytes (<strong c...

Watch this Scientific Journal Video about Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping at JoVE.c

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

isolation-viable-adipocytes-stromal-vascular-fraction-from-human

Research

JoVE Journal

Medicine

3.8K Views.  Instituto Nacional de Perinatologia. Dit protocol biedt een efficiënte collagenase-verteringsmethode voor isolatie van levensvatbare adipocyten en stromale vasculaire fractie-SVF-cellen uit menselijk visceraal vet in één enkel proces, inclusief methodologie om hoogwaardig RNA uit adipocyten te verkrijgen en fenotypificatie van SVF-macrofagen door kleuring van meerdere membraangebonden markers voor analyse door middel van flowcytometrie.

Video: Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Isolation of Adipose Tissue Immune Cells

The discovery of increased macrophage infiltration in the adipose tissue (AT) of obese rodents and humans has led to an intensification of interest in immune cell contribution to local and systemic insulin resistance. Isolation and quantification of different immune cell populations in lean and obese AT is now a commonly utilized technique in immunometabolism laboratories; yet extreme care must be taken both in stromal vascular cell isolation and in the flow cytometry analysis so that the data obtained is reliable and interpretable. In this video we demonstrate how to mince, digest, and isolate the immune cell-enriched stromal vascular fraction. Subsequently, we show how to antibody label macrophages and T lymphocytes and how to properly gate on them in flow cytometry experiments. Representative flow cytometry plots from low fat-fed lean and high fat-fed obese mice are provided. A critical element of this analysis is the use of antibodies that do not fluoresce in channels where AT macrophages are naturally autofluorescent, as well as the use of proper compensation controls.

Adipose tissue (AT) is a site of intense immune cell activation and interaction. Almost all cells of the immune system are present in AT and their ratios are altered by obesity. Proper isolation, quantification, and characterization of AT immune cell populations are critical for understanding their role in immunometabolic disease.

The discovery of increased macrophage infiltration in  the adipose tissue (AT) of obese rodents and humans has led to an  intensification of interest in ...

Immunology and Infection

Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent pathways. Until recently, thermogenic adipocytes were considered a homogeneous population. However, recent studies have indicated that there are multiple subtypes or subpopulations that are distinct in developmental origin, substrate use, and transcriptome. Despite advances in single-cell genomics, unbiased decomposition of adipose tissues into cellular subtypes has been challenging because of the fragile nature of lipid-filled adipocytes. The protocol presented was developed to circumvent these obstacles by effective isolation of single nuclei from adipose tissue for downstream applications, including RNA sequencing. Cellular heterogeneity can then be analyzed by RNA sequencing and bioinformatic analyses.

This publication describes a protocol for the isolation of nuclei from mature adipocytes, purification by fluorescence-activated sorting, and single-cell level transcriptomics.

Brown and beige fat are specialized adipose tissues that dissipate energy for thermogenesis by UCP1 (Uncoupling Protein-1)-dependent and independent ...

Genetics

Isolation and Processing of Murine White Adipocytes for Transcriptome and Epigenome Analyses

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type in white adipose tissue. Understanding how adipocytes function and respond to (epi)genetic and environmental signals is essential for identifying the cause(s) of obesity. RNA and chromatin have previously been isolated from adipocytes using enzymatic digestion. In addition, protocols have been developed for nuclear isolation, where purification is achieved by fluorescence-activated cell sorting (FACS) of adipocyte-specific transgenic reporters. One of the greatest challenges to achieving high yield and quality during such protocols is the substantial amount of lipid contained in adipose tissue. The present protocol describes an optimized procedure for isolating mature adipocytes that leverages heptane to separate lipids from the targets of interest (RNA/chromatin). The resulting RNA has high integrity and generates high-quality RNA-seq results. Likewise, the procedure improves nuclei yield rate and generates reproducible ChIP-seq results across samples. Therefore, the current study provides a reliable and universal murine adipocyte isolation protocol suitable for whole-genome transcriptome and epigenome studies.

The present protocol summarizes a universal method for isolating, purifying, and upstream processing of murine white adipocytes optimized for downstream total RNA sequencing, Nuclei Extraction by SONication (NEXSON), and ChIP-seq.

Obesity is a complex disease influenced by genetics, epigenetics, the environment, and their interactions. Mature adipocytes represent the major cell type ...

Isolation, Expansion, and Adipogenic Induction of CD34+CD31+ Endothelial Cells from Human Omental and Subcutaneous Adipose Tissue

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor response to insulin, local hypoxia, and inflammation. By stimulating the differentiation of functional white adipocytes from progenitors, radical hypertrophy of the adipocyte population can be prevented and, consequently, the metabolic health of adipose tissue can be improved along with a reduction of inflammation. Also, by stimulating a differentiation of beige/brown adipocytes, the total body energy expenditure can be increased, resulting in weight loss. This approach could prevent the development of obesity co-morbidities such as type 2 diabetes and cardiovascular disease.
This paper describes the isolation, expansion, and differentiation of white and beige adipocytes from a subset of human adipose tissue endothelial cells that co-express the CD31 and CD34 markers. The method is relatively cheap and is not labor-intensive. It requires access to human adipose tissue and the subcutaneous depot is suitable for sampling. For this protocol, fresh adipose tissue samples from morbidly obese subjects [body mass index (BMI) &#62;35] are collected during bariatric surgery procedures. Using a sequential immunoseparation from the stromal vascular fraction, enough cells are produced from as little as 2&#8211;3 g of fat. These cells can be expanded in culture over 10&#8211;14 days, can be cryopreserved, and retain their adipogenic properties with passaging up to passage 5&#8211;6. The cells are treated for 14 days with an adipogenic cocktail using a combination of human insulin and the PPAR&#947; agonist-rosiglitazone.
This methodology can be used for obtaining proof of concept experiments on molecular mechanisms that drive adipogenic responses in adipose endothelial cells, or for screening new drugs that can enhance the adipogenic response directed either towards white or beige/brown adipocyte differentiation. Using small subcutaneous biopsies, this methodology can be used to screen out non-responder subjects for clinical trials aimed to stimulate beige/brown and white adipocytes for the treatment of obesity and co-morbidities.

The differentiation of white and beige adipocytes from adipose tissue vascular progenitors bears potential for metabolic improvement in obesity. We describe protocols for a CD34+CD31+ endothelial cell isolation from human fat and for a subsequent in vitro expansion and differentiation into white and beige adipocytes. Several downstream applications are discussed.

Obesity is accompanied by an extensive remodeling of adipose tissue primarily via adipocyte hypertrophy. Extreme adipocyte growth results in a poor ...

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

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

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

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

Biology

Isolation of Adipogenic and Fibro-Inflammatory Stromal Cell Subpopulations from Murine Intra-Abdominal Adipose Depots

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute functionally to the expansion and remodeling of WAT in adulthood. A tremendous barrier to studying the implications of this cellular heterogeneity is the inability to readily isolate functionally distinct cell subpopulations from WAT SVF for in vitro and in vivo analyses. Single-cell sequencing technology has recently identified functionally distinct fibro-inflammatory and adipogenic PDGFR&#946;+ perivascular cell subpopulations in intra-abdominal WAT depots of adult mice. Fibro-inflammatory progenitors (termed, &#8220;FIPs&#8221;) are non-adipogenic collagen producing cells that can exert a pro-inflammatory phenotype. PDGFR&#946;+ adipocyte precursor cells (APCs) are highly adipogenic both in vitro and in vivo upon cell transplantation. Here, we describe multiple methods for the isolation of these stromal cell subpopulations from murine intra-abdominal WAT depots. FIPs and APCs can be isolated by fluorescence-activated cell sorting (FACS) or by taking advantage of biotinylated antibody-based immunomagnetic bead technology. Isolated cells can be used for molecular and functional analysis. Studying the functional properties of stromal cell subpopulation in isolation will expand our current knowledge of adipose tissue remodeling under physiological or pathological conditions on the cellular level.

This protocol describes the technical approach to isolate adipogenic and fibro-inflammatory stromal cell subpopulations from murine intra-abdominal white adipose tissue (WAT) depots by fluorescence-activated cell sorting or immunomagnetic bead separation.

The stromal-vascular fraction (SVF) of white adipose tissue (WAT) is remarkably heterogeneous and consists of numerous cell types that contribute ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.

Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.

In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. ...

Semi-Automated Isolation of Murine White Adipose Tissue

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.

Author Spotlight: Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator  

This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.

The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their ...

Culturing Preadipocytes Isolated from Murine Perivascular Adipose Tissue

Isolation, Culture, and Adipogenic Induction of Stromal Vascular Fraction-derived Preadipocytes from Mouse Periaortic Adipose Tissue

Perivascular adipose tissue (PVAT) is an adipose tissue depot that surrounds blood vessels and exhibits the phenotypes of white, beige, and brown adipocytes. Recent discoveries have shed light on the central role of PVAT in regulating vascular homeostasis and participating in the pathogenesis of cardiovascular diseases. A comprehensive understanding of PVAT properties and regulation is of great importance for the development of future therapies. Primary cultures of periaortic adipocytes are valuable for studying PVAT function and the crosstalk between periaortic adipocytes and vascular cells. This paper presents an economical and feasible protocol for the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, which can be useful for modeling adipogenesis or lipogenesis in vitro. The protocol outlines tissue processing and cell differentiation for culturing periaortic adipocytes from young mice. This protocol will provide the technological cornerstone at the bench side for the investigation of PVAT function.

Author Spotlight: The Significance of Isolation, Culture, and Adipogenic Induction of SVF-Derived Preadipocytes from Mouse Perivascular Adipose Tissue 

Here, we describe the isolation, culture, and adipogenic induction of stromal vascular fraction-derived preadipocytes from mouse periaortic adipose tissue, allowing for the study of perivascular adipose tissue function and its relationship with vascular cells.

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

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