We thank Anke Jurisch, Diana Woellner, Dr. Kathrin Witte and Cornelia Heckmann for assistance with experimental procedures and Benjamin Tiburzy from Biolegend for helpful comments on the gating strategy. J.S. was supported by the Helmholtz Grant (ICEMED). This study was supported by grants from the Clinical Research Unit of the Berlin Institute of Health (BIH), the &#8220;BCRT-grant&#8221; by the German Federal Ministry of Education and Research and the Einstein Foundation. K.S.-B. and H.-D.V. are funded by FOR2165.

This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the Animal Welfare Act under the supervision of our institutional Animal Care and Use Committee. Animal protocols were conducted according to institutional ethical guidelines of the Charit&#233; Berlin, Germany, and were approved by the Landesamt f&#252;r Gesundheit und Soziales and comply with the ARRIVE guidelines.
1. Diet-induced Animal Model of Steatohep...

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The authors have nothing to disclose.

Steatohepatitis has a strong association with metabolic abnormalities such as obesity, insulin resistance and dyslipidemia15. Multiple studies indicate that adipose tissue inflammation can drive the pathogenesis of type 2 diabetes, including altered levels of cells of both the innate and adaptive immune system4,5,16,17. In addition, it has been found that obesity modulat...

Obesity is a multifactorial disorder and a major risk factor for developing heart disease, stroke, nonalcoholic steatohepatitis (NASH), type 2 diabetes (T2D) and some types of cancer. The prevalence of obesity is rapidly increasing globally. Today, 2.1 billion people &#8212;&#160;nearly 30% of the world&#8217;s population &#8212; are either obese or overweight1. Obesity-associated insulin resistance can lead to T2D, when exhausted pancreatic islet beta cells fail to compensate for the increased need for insulin to maintain glucose homeostasis2.
Adipose tissue is composed of various cell types including adipocytes, endothelial cells, fibroblasts and immune cells. During progression of obesity, changes in the number and activity of immune cells can lead to low-grade inflammation of hypertrophic adipose tissue3,4. Specifically, it has been found that excessive energy intake, accompanied by chronically elevated levels of blood glucose, triglycerides and free fatty acids, leads to adipocyte hypoxia, endoplasmic reticulum stress, impaired mitochondrial function and enhanced cytokine secretion, resulting in the activation of pro-inflammatory adipose immune cells5,6. Past research has mainly focused on innate immunity, but more recently adaptive immune cells (T and B cells) have emerged as important regulators of glucose homeostasis. They possess inflammatory (including CD8+ T cells, Th1, and B cells) or primarily regulatory functions (including regulatory T (Treg) cells, Th2 cells) and can both exacerbate or protect against insulin resistance7,8,9.
Furthermore, several mechanisms were proposed to explain how obesity increases steatohepatitis, including increased production of cytokines by adipose tissue10. NASH, the progressive form of nonalcoholic fatty liver disease and a major health burden in developed countries, is histologically characterized by ballooned hepatocytes, lipid accumulation, fibrosis and pericellular inflammation and may progress to cirrhosis, end stage liver disease or hepatocellular carcinoma. Several regimen (for instance the methionine and choline deficient diet11) are known to induce NASH-like liver pathology in non-human animal models, but most of these approaches do not recapitulate human conditions of NASH and its metabolic consequences as they either require specific gene knockout, non-physiological dietary manipulations or lack insulin resistance typical of human NASH. Moreover, our understanding of the underlying mechanisms of metabolic diseases is currently based on experiments carried out with laboratory mice housed under standard specific pathogen free (SPF) conditions. Those barrier facilities are abnormally hygienic and do not consider the microbial diversity humans have to encounter, which may account for difficulties in the translation process of animal studies to clinical approaches12,13,14.
To investigate the different immune cell subsets in adipose tissue and liver during the development of insulin resistance and NASH in an advanced mouse model reproducing human immunological conditions, mice were housed in individual cages in semi sterile conditions without a barrier. Mice housed under antigen exposed conditions developed NASH-like liver pathology already after 15 weeks of high-fat diet (HFD) feeding13. Compared to age-matched SPF mice they developed macrovesicular steatosis, hepatic infiltration and activation of immune cells.
This manuscript describes a robust flow cytometry analysis to define and count immune cell subsets from mouse adipose tissue and liver in a model of NASH. Flow cytometry analysis allows the detection of multiple parameters of individual cells simultaneously in contrast to RT-PCR or immunohistochemistry approaches.
In summary, our study offers a mouse model of short-term HFD for investigating the development of insulin resistance and NASH and the underlying mechanisms that also exhibits fidelity to the human condition.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">100&#181;m cell strainers&#160;</td>
			<td style="width:169px;">Falcon</td>
			<td style="width:119px;">352340</td>
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			<td height="21" style="height:21px;width:351px;">1ml syringe&#160;</td>
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			<td style="width:119px;">309659</td>
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			<td height="21" style="height:21px;width:351px;">26G x 5/8 needles&#160;</td>
			<td style="width:169px;">BD&#160;</td>
			<td style="width:119px;">305115</td>
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			<td height="21" style="height:21px;width:351px;">35mm Petri Dishes&#160;</td>
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			<td style="width:169px;">Falcon</td>
			<td style="width:119px;">352340</td>
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			<td height="21" style="height:21px;width:351px;">ACK lysis buffer&#160;</td>
			<td style="width:169px;">GIBCO</td>
			<td>A1049201</td>
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			<td height="21" style="height:21px;width:351px;">Alexa Fluor 700 anti-mouse CD45</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">103127</td>
			<td>AB_493714 (BioLegend Cat. No. 103127)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Analysis software&#160;</td>
			<td>FlowJo 10.0.8 software</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">APC anti-mouse CD11c Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">117309</td>
			<td>AB_313778 (BioLegend Cat. No. 117309)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">APC anti-mouse KLRG1 (MAFA) Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">138411</td>
			<td>AB_10645509 (BioLegend Cat. No. 138411)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV421 anti-mouse CD127 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">135023</td>
			<td>AB_10897948 (BioLegend Cat. No. 135023)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV421 anti-mouse F4/80 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">123131</td>
			<td>AB_10901171 (BioLegend Cat. No. 123131)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV605 anti-mouse CD279 (PD-1) Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">135219</td>
			<td>AB_11125371 (BioLegend Cat. No. 135219)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV605 anti-mouse NK-1.1 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">108739</td>
			<td>AB_2562273 (BioLegend Cat. No. 108739)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV650 anti-mouse/human CD11b Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">101239</td>
			<td>AB_11125575 (BioLegend Cat. No. 101239)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV711 anti-mouse/human B220 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">103255</td>
			<td>AB_2563491 (BioLegend Cat. No. 103255)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">BV785 anti-mouse CD8a Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">100749</td>
			<td>AB_11218801 (BioLegend Cat. No. 100749)</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:39px;width:351px;">C57Bl/6J mice, male, 5 weeks old&#160;</td>
			<td style="width:169px;">Forschungseinrichtungen f&#252;r experimentelle Medizin (FEM)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">CaCl2&#160;</td>
			<td>Charit&#233; - Universit&#228;tsmedizin Berlin</td>
			<td style="width:119px;">A119.1&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:351px;">Collagenase NB 4G Proved Grade&#160;</td>
			<td style="width:169px;">SERVA&#160;</td>
			<td style="width:119px;">11427513</td>
			<td></td>
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			<td height="36" style="height:36px;width:351px;">Collagenase Typ I&#160;</td>
			<td style="width:169px;">Worthington&#160;</td>
			<td style="width:119px;">LS004197</td>
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			<td height="21" style="height:21px;width:351px;">Conical centrifuge tube 15ml&#160;</td>
			<td style="width:169px;">Falcon</td>
			<td style="width:119px;">352096</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Conical centrifuge tube 50ml&#160;</td>
			<td style="width:169px;">Falcon</td>
			<td style="width:119px;">352070</td>
			<td></td>
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			<td height="21" style="height:21px;width:351px;">DNAse&#160;&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">4716728001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Fetal bovine serum&#160;</td>
			<td style="width:169px;">Biochrom</td>
			<td style="width:119px;">S0115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Filter 30&#181;m&#160;</td>
			<td style="width:169px;">Celltrics&#160;</td>
			<td style="width:119px;">400422316</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">FITC anti-mouse CD3 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">100203</td>
			<td>AB_312660 (BioLegend Cat. No. 100203)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Flow cytometry&#160;</td>
			<td style="width:169px;">BD-LSR Fortessa&#160;</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Forceps&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td>F4142-1EA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">HBSS&#160;</td>
			<td style="width:169px;">Bioanalytic GmBH&#160;</td>
			<td style="width:119px;">085021-0500&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">High-fat diet&#160;</td>
			<td style="width:169px;">SSNIF</td>
			<td>E15741&#8211;34&#160;</td>
			<td>60 kJ% from fat, 19&#8201;kJ% from proteins, and 21&#8201;kJ% from carbohydrates</td>
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			<td height="21" style="height:21px;width:351px;">micro dissecting scissors&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">S3146</td>
			<td>used for dissection purposes&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PE anti-mouse CD25 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">101903</td>
			<td>AB_312846 (BioLegend Cat. No. 101903)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PE/Cy7 anti-mouse CD62L Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">104417</td>
			<td>AB_313102 (BioLegend Cat. No. 104417)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PE/Cy7 anti-mouse I-A/I-E (MHCII) Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">107629</td>
			<td>AB_2290801 (BioLegend Cat. No. 107629)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PE/Dazzle 594 anti-mouse CD4 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">100565</td>
			<td>AB_2563684 (BioLegend Cat. No. 100565)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Percoll solution&#160;</td>
			<td style="width:169px;">Biochrom</td>
			<td style="width:119px;">L6115</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PerCP/Cy5.5 anti-mouse CD44 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">103031</td>
			<td>AB_2076206 (BioLegend Cat. No. 103031)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">PerCP/Cy5.5 anti-mouse Gr-1 Antibody</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">108427</td>
			<td>AB_893561 (BioLegend Cat. No. 108427)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Phosphate buffered saline&#160;</td>
			<td style="width:169px;">Gibco</td>
			<td style="width:119px;">12559069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Round-Bottom Tubes with cell strainer cap</td>
			<td style="width:169px;">STEMCELL Technologies&#160;</td>
			<td style="width:119px;">38030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">TruStain fcX anti-mouse CD16/32</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">101301</td>
			<td>AB_312800 (BioLegend Cat. No. 101301)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Trypan Blue&#160;</td>
			<td style="width:169px;">Sigma-Aldrich&#160;</td>
			<td style="width:119px;">T6146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Zombie NIR Fixable Viability Kit</td>
			<td style="width:169px;">Biolegend&#160;</td>
			<td style="width:119px;">423105</td>
			<td>viablity stain&#160;</td>
		</tr>
	</tbody>
</table>

an advanced murine model for nonalcoholic steatohepatitis in association with type 2 diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

The protocol described allows the characterization of surface markers of innate and adaptive immune cells isolated from murine perigonadal adipose tissue and liver in a model of diet-induced NASH. In this model, NASH was induced by administration of HFD plus sucrose (6%) in drinking water for 7 to 15 weeks in C57Bl/6J mice, as previously reported13. Importantly, mice were housed in semi sterile conditions and, thus, exposed to environmental antigens throughout the experiment. HFD fed mice housed i...

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An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

This method can help answer questions in the immunometabolism field about the contribution of immune cells to obesity-induced inflammation in adipose and steatohepatitis in hepatic tissue. These techniques have been optimized for the isolation of adipose and hepatic immune cells in a diet-induced model of nonalcoholic steatohepatitis achieved through a non-specific pathogen-free rodent housing. These methods can help to deepen our understanding of the immunological mechanisms involved in the maintenance of metabolic homeostasis.Generally, individuals new to this technique will struggle with maximizing the yield of functionally viable dissociated cells and setting up the proper gating in flow cytometry. To generate an adipose tissue single-cell suspension, spray the chest of a euthanized mouse with 70%ethanol, and carefully make a five to six-centimeter central incision through the integument and abdominal wall along the entire length of the rib cage to expose the pleural cavity and heart. Using a 26-gauge needle, inject at least 10 milliliters of 0.9%saline solution into the apex of the left ventricle, and use scissors to open the peritoneal cavity.Then, dissect the epigonadal adipose tissue, and weigh it. Mechanically dissociate the adipose tissue into fine pieces in a Petri dish at four degrees Celsius before transferring the tissue fragments into a 50-milliliter conical tube. Rinse the Petri dish with one milliliter of 0.5%bovine serum albumin, or BSA, in PBS, and add three milliliters of freshly prepared adipose tissue digest solution per gram of adipose tissue to the tube.After 20 minutes at 37 degrees Celsius and 200 rotations per minute, add five milliliters of 0.5%BSA in PBS, and place the adipose digest on ice. Triturate the solution multiple times with a 10-milliliter serological pipette, and use a plunger to pass the solution through a 100-micrometer strainer. Separate the tissue fractions by centrifugation, and use a pipette to discard the floating adipocyte fraction.Resuspend the stromal vascular fraction cell pellet in one milliliter of ammonium-chloride-potassium, or ACK, lysis buffer, stopping the lysis reaction with 10 milliliters of 2%fetal calf serum, or FCS, in PBS after a few seconds. Then, collect the isolated white blood cells by centrifugation, and resuspend the pellet in 250 microliters of 2%FCS in PBS for counting. Harvest the liver into a conical tube of PBS on ice before using syringe stamps to mechanically dissociate the tissue in a Petri dish at four degrees Celsius.Transfer the dissected liver tissue fragments into a 50-milliliter tube containing 10 milliliters of warm liver digest solution, and rinse the Petri dish with three milliliters of fresh liver digest solution. After 20 minutes at 37 degrees Celsius and 200 rotations per minute, stop the digestion with 20 milliliters of HBSS, and triturate the tissue slurry multiple times with a 10-milliliter serological pipette. Use a plunger to pass the tissue solution through a 100-micrometer strainer, and collect the liver cells by centrifugation.Resuspend the pellet in 20 milliliters of fresh HBSS, and remove the hepatocyte matrix by centrifugation. Collect the supernatant for centrifugation, resuspending the pellet in 10 milliliters of 33%low viscosity density gradient medium solution in HBSS. Separate the liver immune cells by density gradient centrifugation, and resuspend the leukocyte pellet in one milliliter of ACK lysis buffer.After four minutes at room temperature, stop the lysis with 10 milliliters of HBSS, and filter the cells through a 30-micrometer pore strainer into a 15-milliliter tube for centrifugation. Then, resuspend the pellet in 250 microliters of 2%FCS in PBS for counting. For flow cytometric analysis of the isolated leukocyte populations, add up to three times 10 to the six cells in 100 microliters of 2%FCS in PBS from the tissue of interest into individual polystyrene fluorescence-activated cell sorting, or FACS, tubes.Add 10 microliters of anti-CD16/CD32 antibody to each tube to block any non-specific binding for 10 minutes on ice, followed by staining with the appropriate volume of antibody mixture and one microliter of viability dye per sample to allow live and dead cell discrimination. After 20 minutes at four degrees Celsius protected from light, wash the samples two times with two milliliters of 2%FCS plus PBS per wash, and centrifuge at 300 times g for five minutes at four degrees Celsius. Then, resuspend the pellet in fresh PBS plus FCS.To analyze the cells by flow cytometry, use an unstained negative control sample to set the forward and side scatter, and adjust the voltages of the flow cytometer to detect the viable leukocyte population and to exclude the debris. Next, run single stained control samples for multicolor compensation before running the experimental samples, collecting at least five times 10 to the four events per tube. Then, export the FCS data files for analysis, and set the gating strategy for CD45-positive leukocytes to allow the identification of the subsequent immune cell populations of interest.Here, a representative gating strategy for T cell subpopulations including doublet discrimination and viability staining in murine perigonadal fat is shown. CD45-positive leukocytes are first gated for CD4 and CD8 and subsequently for CD44 and CD62 ligand to discriminate between naive, central memory, effector memory, and effector T cells. CD44-positive cells are then further characterized with CD127, killer cell lectin-like receptor G1, and programmed death-1.Here, the gating strategy for analyzing B cells, granulocytes, natural killer cells, macrophages, and dendritic cells is shown. A higher percentage of effector memory CD4-positive and CD8-positive cells can be detected in high-fat diet mice housed in non-SPF conditions, whereas intrahepatic naive CD4-positive and CD8-positive T cells are found to be considerably lower in these animals compared to SPF high-fat diet mice at week seven. Severe steatosis, including an increase of large lipid droplets resulting in macrovesicular steatosis, lobular inflammation, hepatocellular ballooning, and destroyed lobular structure is found in high-fat diet exposed mice while only some SPF mice display a mild fat accumulation in the liver.In addition, the percentage of natural killer cells is higher in the perigonadal adipose tissue of high-fat diet exposed mice, whereas high-fat diet SPF mice demonstrated higher percentages of dendritic cells. When attempting this procedure, it is important to keep the cells on ice and in dark unless otherwise noted. You should now have a good understanding of how to properly prepare single-cell suspensions and how to isolate murine adipose and hepatic tissue for flow cytometry experiments.

an-advanced-murine-model-for-nonalcoholic-steatohepatitis-association

Research

JoVE Journal

Immunology and Infection

6.9K Görüntüleme.  Humboldt-Universität zu Berlin. Bu yöntem, immünmetabolizma alanındaki immünmetabolizma alanındaki soruların yanıtlanarak, immün hücrelerin obeziteye bağlı inflamasyona katkısı ve karaciğer dokusunda steatohepatit yardımcı olabilir. Bu teknikler non-spesifik patojen içermeyen kemirgen muhafaza yoluyla elde edilen nonalkolik steatohepatit diyet kaynaklı bir model yağ ve hepatik bağışıklık hücrelerinin izolasyon uğrup için optimize edilmiştir. Bu yöntemler metabolik homeostazın bakımında rol oy...