Akış Sitometresi çekirdek tesis Pennsylvania State Üniversitesi Millennium bilim Complex adlı teşekkür etmek istiyorum.

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	<li>Davies, L. C., Taylor, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages:+Then+and+Now.">Tissue-resident macrophages: Then and Now.</a> Immunology. 144 (4), 541-548 (2015).</li><li>Kahn, S. E., Hull, R. L., Utzschneider, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+linking+obesity+to+insulin+resistance+and+type+2+diabetes.">Mechanisms linking obesity to insulin resistance and type 2 diabetes.</a> Nature. 444 (12), 840-846 (2006).</li><li>Van Gaal, L. F., Mertens, I. L., De Block, C. 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Yazarlar ifşa etmek yok çatışması var.

Komorbiditeler ateroskleroz, basit yağlanma, steatohepatitis gibi taklit ve tip 2 diyabet diyet kaynaklı metabolik bozukluk modelleri temel moleküler mekanizmaları hastalık progresyon daha iyi anlamak için yaygın olarak kullanılır. Collagenase bağımlı sindirim genellikle doku hücreleri hücre dışı Matriks (ECM)16,27den kurtarmak için ayırmak için kullanılır. Collagenase gibi enzimler hücre komşu için yapısal destek sağlayan kolajen bozab...

Fare modelleri yoğun insan hastalıkları dinamiklerini incelemek için kullanılmaktadır. Fareler hastalıklı bir durumda doku yerleşik hücre uygun yalıtım Patogenez moleküler ve hücresel katkılar hastalığı1anlamak için bir platform sağlar. Kritik önemi olan bir obezite bozukluğudur. Obezite sıklığı 2 diyabet, kalp-damar hastalıkları ve yağlı karaciğer hastalığı2,3yazın ve insülin direnci ile paralel olarak dünya çapında yükselmeye devam ediyor. Aşırı besin tüketimi daha da azalmış fiziksel aktivite olan aort ve karaciğer4gibi diğer periferik dokuların hücresel ortamı değiştirmek Yağ dokusundan kaynaklanan değişmiş sinyalleri tetikleyen tarafından çarpık. Bu tür bozulma metabolik homeostazı sonuçları bir kronik düşük dereceli sistemik inflamasyon5.
Klasik makrofajlar aorta ve karaciğer gibi beyaz Yağ dokusundan (WAT) onların işe alım için ikamet aktivasyonu sadece bozukluk metabolik sinyallerin başlatmak ama aynı zamanda iltihabı6,7sürdürmek için gösterilmiştir. Makrofajlar fenotipik ve fonksiyonel heterojen şiddetle obezite patogenezi ile ilişkilidir ilgili komorbiditeler7. Makrofaj polarizasyon dinamik plastisite bu hücreler bir dizi ilerleme koordine aktif fenotipleri ve inflamasyon8çözünürlüğe sergilemek sağlar. Klasik aktif (M1) makrofajlar iltihap yayılmasında karıştığı iken, alternatif olarak aktif (M2) makrofajlar çözünürlük ve doku onarımı9,10ile ilişkilendirilmiştir.
Vücudun metabolik stres uğrar gibi beyaz yağ dokusu anormal derecede birikir. Genişletilmiş yağ dokusu çeker ve derinden insülin direnci, hiperglisemi ve sonuçta tip 2 diabetes mellitus, insülin direnci veya hiperglisemi11tanıtmak için normal adipocyte işlevini değiştirme inflamatuar hücreleri korur, 12. Buna paralel olarak, beyaz yağ dokusu değişikler yanıt inflamatuar sinyalleri tarafından yayımlanan olarak klasik aktif (M1) yağ doku makrofajlar (TYS)13,14sızmış. Çok hücreli bu organ aort ve karaciğer4gibi diğer vücut organlarının normal fonksiyon raydan çıktı bir çağlayan sinyal yayıyor.
Karaciğer yakındaki dysregulated WAT15kaynaklanan uyaranlara yanıt olarak uyum sağlar metabolik bir elektrik santrali var. Hepatik makrofajlar veya Kupffer hücreleri, metabolik değişiklikler, hem parenkima dönüşümü inflamatuar sitokinlerin ve parenkimal olmayan hücre fenotip salgılar ve doku remodeling teşvik. Karaciğer lipid birikimi, iltihap, aşırı hücre dışı matriks mevduat, nekroz ve karaciğer hasarı geniş yelpazede katkıda inflamatuar hakaret non alkolik yağlı karaciğer hastalığı ile ilgili nihai işlev kaybı aşağıdaki gibidir 16,17,18.
Vücudun kronik metabolik stres19uğrar gibi güvenliği aşılan WAT ve karaciğer fonksiyon için paralel olarak büyük arterlerin lipidler Arteryel duvardaki içinde birikir. Arteryel lipid birikimi kemokinler aktive endotel hücreleri tarafından salgılanmasını ve monosit20sonraki işe alım tetikler. Bir kez işe, monosit çoğalırlar, ayırt etmek, lipoproteinler yutmayın ve köpük hücreleri haline. Atherogenesis başlatılan ve pro-inflamatuar etkinliği tarafından sürekli işe ve doku ikamet lipid yüklü makrofajlar. Bu aterojenik microenvironment geçirilen ekstraselüler ve hücre içi stres sinyallerini succumbing, bu makrofajlar sonra art arda sıralı sinyal bir apoptotik meşgul. Bu köpük hücreleri ölürken dolu lipid içeriklerini daha sonra plak rüptürü, miyokard enfarktüsü ve inme için neden lezyon nekrotik çekirdeğini katkıda bulunur.
Topluca, makrofaj fenotipleri heterojen kısmen enflamatuar degisimler WAT, karaciğer ve aort8,21gibi dysregulated dokularda gözlenen tarafından indüklenen obezite orchestrates. Karakterizasyonu işe ve ikamet makrofajlar doku makrofaj fenotip1işlemek potansiyel moleküler hedeflerin içgörü sağlayabilir. Etkili bir şekilde gelen obezite kaynaklı iltihaplı doku makrofajlar karakterize etmek için bir tek hücre süspansiyon Enzimatik sindirim elde edilebilir. Böyle ayrılma iletişim kurallarının yeterince bağ dokusu bağışıklık hücre ölümü en aza indirilmesi ve optimum hücre verim sağlayan aşağılayıcı içinde etkili olması gerekir. Enzim karışımı doku ve yapısal onun makyaj türüne bağlıdır. Aort damarı gibi esnek doku karaciğer ve WAT doku ayrılma elde etmek için kıyasla daha güçlü enzimatik aktivite gerektirir. Tek hücre süspansiyon, doku ikamet makrofajlar olabilir belirsizliğe yer bırakmadan ile karakterize veya transkripsiyon profil oluşturma gibi daha da aşağı akım analizleri için izole.
Burada bir doku özel iletişim kuralı etkin bir şekilde izole ve doku yerleşik makrofajlar indüklenen geleneksel beslenme obezite elde edilen karakterize için collagenase bağlı doku sindirim ve renkliden Akış Sitometresi kullanan anlatılan, ateroskleroz, basit yağlanma ve steatohepatitis fare modelleri. Eşzamanlı lökosit-(CD45 ve/veya CD11b) ve makrofaj - karşı antikor ile hücre yüzey işaretlerinin boyama (F4/80) belirli antijenleri kez makrofaj nüfus22tanımlamak için kullanılır. Floresans aktif hücre (FACS) sıralama yüksek saflıkta, saptanan bu nüfus sıralamak için kullanılan güçlü bir stratejidir. Sıralanmış nüfus daha sonra aşağı akım moleküler analizi (gibi nicel gerçek zamanlı Polimeraz zincir reaksiyonu)23kullanarak fenotip belirli bir gen profilleri için değerlendirilebilir. Standart Akış Sitometresi ve Akış Sitometresi tabanlı cep sıralama makrofajlar içinde büyük ölçüde türdeş olmayan hücre süspansiyon ayrım içinde güçlü araçlar olmakla birlikte, eski protokoller başarılı çıkış sağlamak için optimize edilmiş olması gerekir. Bu çalışmada, etkili bir şekilde izole etmek ve uygun doku belirli makrofajlar karakterize protokolleri açıklanmıştır; daha da önemlisi, bu çalışmada, önlemek ve/veya bunları çözmek için proaktif ve LNB'ler stratejileri yanı sıra sık sık ortaya çıkan teknik sorunları içine çok önemli bilgiler sağlar.

<table><tbody><tr><td>26 G x 5/8 in Needles</td><td>BD</td><td>305115</td><td></td></tr><tr><td>23 G x 0.75 in needle x 12 in. tubing Blood Collection Set</td><td>BD</td><td>367297</td><td>Used for cannulation of subhepatic IVC during liver perfusion</td></tr><tr><td>21 G 1 1/2 in. Needles</td><td>BD</td><td>305156</td><td></td></tr><tr><td>1 mL syringe with rubber stops</td><td>BD</td><td>309659</td><td></td></tr><tr><td>10 mL Syringes</td><td>BD</td><td>309604</td><td></td></tr><tr><td>1 mL Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>F4/80 PE</td><td>Biolegend</td><td>123110</td><td></td></tr><tr><td>CD11c PE/Cy7</td><td>Biolegend</td><td>117318</td><td></td></tr><tr><td>CD11b PE/Cy5</td><td>eBioscience</td><td>15-0112-81</td><td></td></tr><tr><td>Anti-mouse CD16/32 Fc Block&amp;nbsp;</td><td>Biolegend</td><td>101320</td><td></td></tr><tr><td>CD45 Pacific Blue</td><td>Biolegend</td><td>103126</td><td></td></tr><tr><td>PE Rat IgG2a</td><td>Biolegend</td><td>400508</td><td></td></tr><tr><td>PE/Cy7 Armenian Hamster IgG</td><td>Biolegend</td><td>400922</td><td></td></tr><tr><td>PE/Cy5 Rat IgG2b</td><td>Biolegend</td><td>400610</td><td></td></tr><tr><td>Pacific Blue Rat IgG2c</td><td>Biolegend</td><td>400717</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)&amp;nbsp;</td><td>Cellgro</td><td>15-017-CV</td><td></td></tr><tr><td>1x Phosphate Buffered Saline (PBS)</td><td>Cellgro</td><td>21-031-CV</td><td></td></tr><tr><td>70 &amp;mu;m cell strainers</td><td>Corning, Inc.</td><td>352350</td><td></td></tr><tr><td>1.7 mL microcentrifuge tube&amp;nbsp;</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Paraformaldehyde Aqueous Solution -16x</td><td>Electron Microscopy Sciences</td><td>CAS #30525-89-4</td><td></td></tr><tr><td>Micro Dissecting Scissors, 3.5 inch, Straight, 23 mm, Sharp&amp;nbsp;</td><td>Stoelting</td><td>52132-10P</td><td>Used for general dissecting purposes</td></tr><tr><td>Micro Forceps, 4 inches, full curve, 0.8 mm</td><td>Stoelting</td><td>52102-37P&amp;nbsp;</td><td>Used for general dissecting purposes</td></tr><tr><td>Spring dissection scissors &amp;ndash; 3 mm Cutting edge&amp;nbsp;</td><td>Fine Science Tool</td><td>15000-10</td><td>Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td></tr><tr><td>Curved 0.07 mm x 0.04&amp;nbsp;mm Tip Forceps</td><td>Fine Science Tool</td><td>11297-10</td><td>Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td></tr><tr><td>Hemostatic Forceps (Curved)</td><td>Fine Science Tool</td><td>13021-12</td><td></td></tr><tr><td>Heparin Sodium Salt</td><td>Fischer Scientific</td><td>9041-08-1</td><td></td></tr><tr><td>35 mm Cell Culture/Petri Dishes</td><td>Fischer Scientific</td><td>12-565-90</td><td></td></tr><tr><td>Polystyrene Petri Dishes (10 cm) w/lid</td><td>Fischer Scientific</td><td>08-757-100D</td><td></td></tr><tr><td>15 mL Conical Centrifuge Tubes (Polypropylene)</td><td>Fischer Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>50 mL Conical Centrifuge Tubes&amp;nbsp;(Polypropylene)</td><td>Fischer Scientific</td><td>14-432-22</td><td></td></tr><tr><td>5 mL Round-Bottom Polystyrene Tubes&amp;nbsp;</td><td>Fischer Scientific</td><td>14-959-5</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gemini Bio-Products</td><td>100-106</td><td></td></tr><tr><td>Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous)</td><td>Millipore</td><td>EX0285-1</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Rockland</td><td>BSA-50</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Collagenase Type II</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>Collagenasse Type XI</td><td>Sigma-Aldrich</td><td>C7657</td><td></td></tr><tr><td>Hyaluronidase Type I</td><td>Sigma-Aldrich</td><td>H3506</td><td></td></tr><tr><td>DNAse</td><td>Sigma-Aldrich</td><td>DN25</td><td></td></tr><tr><td>Collagenase Type I</td><td>Sigma-Aldrich</td><td>C0130</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>1310-73-2&amp;nbsp;</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma Aldrich</td><td>449709-10G</td><td></td></tr><tr><td>500 mL beaker&amp;nbsp;</td><td>Sigma Aldrich</td><td>02-540M</td><td></td></tr><tr><td>4 cm Hemostatic clamp</td><td>Stoelting</td><td>52120-40</td><td></td></tr><tr><td>Toothed forceps</td><td>Stoelting</td><td>52104-33P</td><td></td></tr><tr><td>50 &amp;mu;m Disposable filters</td><td>Systemex</td><td>04-0042-2317</td><td></td></tr><tr><td>Collagenase Type IV</td><td>ThermoFischer Scientific</td><td>17104019</td><td></td></tr><tr><td>Ammonium Chloride Potassium (ACK)</td><td>ThermoFischer Scientific&amp;nbsp;</td><td>A1049201</td><td></td></tr><tr><td>Razors (0.22 mm (0.009&quot;))</td><td>VWR International</td><td>55411-050</td><td></td></tr><tr><td>Texas Red Live/Dead stain</td><td></td><td></td><td>Red viability stain (in &lt;strong&gt;Figure 1A&lt;/strong&gt;)</td></tr></tbody></table>

isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation

Obezite büyük ölçüde doku yerleşik makrofajlar gibi monosit kaynaklı makrofajlar tarafından aracılık ettiği kronik bir enflamatuar devlet teşvik etmektedir. Diyet kaynaklı obezite (DIO) makrofaj heterojenite rolü eğitim değerli bir modeldir; Ancak, yeterli makrofaj izolasyonların iltihaplı dokulara elde etmek zordur. Bu protokol için biz yalıtım adımları ve doku yerleşik makrofajlar uygun nüfusu yüksek yağlı (HFD) veya yüksek yağ/yüksek kolesterol (18 hafta takip fareler elde etmek için çalışmalarımız elde gerekli hata giderme kılavuzu anahat HFHCD) diyet müdahale. Bu iletişim kuralı obezite ve karaciğer, beyaz yağ dokuları (WAT) ve aort damarı da dahil olmak üzere ateroskleroz okudu üç hallmark dokular üzerinde duruluyor. Biz nasıl ikici kullanımı vurgulayın Akış Sitometresi yalıtım yeni bir boyut ve doku yerleşik makrofajlar karakterizasyonu elde edebilirsiniz. Bu iletişim kuralının temel bir bölümünü doku özgü Enzimatik sindirim ve makrofaj yalıtım ve akış sitometrik çözümlemesi için boyama sonraki hücre yüzeyine antikor temel inceliklerini giderir. Bu iletişim kuralı mevcut karmaşıklığı floresan aktif hücre (FACS) sıralama temel giderir ve geniş karakterizasyon yeterli sıralanmış hücre gruplarından elde etmek için bu karmaşıklığı için açıklamalar sunar. Alternatif zenginleştirme yöntemleri gibi FACS ile çalışırken esneklik ve zaman yönetimi için izin yoğun karaciğer hücreleri sıralamak için dahil edilir. Kısacası, bu iletişim kuralı çok sayıda belirli bir çalışma içinde iltihaplı doku makrofaj heterojenite değerlendirmek için araştırmacı yardımcıları ve olumlu hücresel yalıtım ve karakterizasyonu için başarılı olmuştur anlayışlı sorun giderme ipuçları sağlar iltihap DIO-aracılı bağışıklık hücrelerinin.

Apolipoprotein E eksik (ApoE KO) C57BL/6 (BL6) fareler yüksek yağ yüksek kolesterol diyet (HCHFD veya HCD) 18 hafta, 1 x 104 - 2 x 104 CD45 tutulan kullanırken+F4/80+ ne zaman iki örnek aortik makrofajlar izole olabilir havuza alınmış. HFHCD beslenen ApoE KO fareler disseke ciğeri üretilen (ki mevcut sıralama zaman bağlıdır) 5 x 10 göre5 Kupffer hücreleri daha büyük. Ne zaman yüksek yağlı diyet (HFD) kullanarak be...

Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Bu iletişim kuralı ayırmak ve karakterize bir araştırmacı doku yerleşik sağlar makrofajlar içinde çeşitli metabolik bozuklukların diyet kaynaklı modellerinden çıkarılan iltihaplı dokulara hallmark.

Yalıtım, karakterizasyonu ve obezite ile ilgili iltihap tarafından etkilenen dokular makrofajlar arıtma

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

isolation-characterization-purification-macrophages-from-tissues

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

25.1K Görüntüleme.  The Pennsylvania State University. Bu iletişim kuralı ayırmak ve karakterize bir araştırmacı doku yerleşik sağlar makrofajlar içinde çeşitli metabolik bozuklukların diyet kaynaklı modellerinden çıkarılan iltihaplı dokulara hallmark.

Video: Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Yetişkin Karaciğer Hücrelerinin Karma İlköğretim Kültürler Makrofajlar yalıtın Basit ve Etkin Yöntem

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

İmmünoloji ve Enfeksiyon

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

Düşük oksijen ortamı altında insan makrofaj polarizasyon proteomik analizi

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

Biyokimya

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

Kemik İliği Türetilmiş makrofajlar kullanarak Makrofaj Polarizasyon İncelenmesi

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

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.

Yağ Doku Bağışıklık Hücre İzolasyonu

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

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

İnsan Çift Gradient Santrifüj ile Monositlerinde ve teflon kaplı Hücre Kültürü Çantalar Makrofagların Onların Farklılaşma İzolasyonu

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. Macrophages express surface Toll-like receptors (TLRs), which recognize molecular patterns conserved through evolution in a wide range of microorganisms. TLRs play a central role in macrophage activation which is usually associated with gene expression alteration. Macrophages are critical in many diseases and have emerged as attractive targets for therapy. In the following protocol, we describe a procedure to isolate murine peritoneal macrophages using Brewer&#8217;s thioglycollate medium. The latter will boost monocyte migration into the peritoneum, accordingly this will raise macrophage yield by 10-fold. Several studies have been carried out using bone marrow, spleen or peritoneal derived macrophages. However, peritoneal macrophages were shown to be more mature upon isolation and are more stable in their functionality and phenotype. Thus, macrophages isolated from murine peritoneal cavity present an important cell population that can serve in different immunological and metabolic studies. Once isolated, macrophages were stimulated with different TLR ligands and consequently&#160;gene expression was evaluated.

We describe here a simple protocol to isolate murine peritoneal macrophages. This procedure is followed by RNA extraction to carry out gene expression analysis upon Toll-like receptors stimulation.

Fare Periton Makrofagların izolasyonu Toll-benzeri reseptörler Uyarım üzerine Gen İfadesi Analizi Yapacak

During infection and inflammation, circulating monocytes leave the bloodstream and migrate into tissues, where they differentiate into macrophages. ...

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

In Vitro Phagocytosis of Myelin Debris by Bone Marrow-Derived Macrophages

We present methods to assess the phagocytic capacity of primary murine bone marrow-derived macrophages using fluorescently labeled myelin debris and intracellular lipid droplet staining.

Vitro Fagositoz miyelin enkaz kemik iliği türevi makrofajlar tarafından

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a ...

Nörobilim

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Akış Sitometresi kullanarak insan yağ dokusu içinde bağışıklık hücreleri karakterizasyonu

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Tıp

A Comprehensive High-Efficiency Protocol for Isolation, Culture, Polarization, and Glycolytic Characterization of Bone Marrow-Derived Macrophages

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. Macrophages are commonly classified as M1-like (inflammatory) and M2-like (anti-inflammatory) subtypes. M1-like macrophages are pro-inflammatory macrophages that get activated by LPS and/or pro-inflammatory cytokines such as INF-&#947;, IL-12 &#38; IL-2. M1-like polarized macrophages are involved in various diseases by mediating the host&#39;s defense to a variety of bacteria and viruses. That is very important to study LPS induced M1-like macrophages and their metabolic states in inflammatory diseases. M2-like macrophages are considered anti-inflammatory macrophages, activated by anti-inflammatory cytokines and stimulators. Under the pro-inflammatory state, macrophages show increased glycolysis in glycolytic function. The glycolytic function has been actively investigated in the context of glycolysis, glycolytic capacity, glycolytic reserve, compensatory glycolysis, or non-glycolytic acidification using extracellular flux (XF) analyzers.
This paper demonstrates how to assess the glycolytic states in real-time with easy-to-follow steps when the bone marrow-derived macrophages (BMDMs) are respiring, consuming, and producing energy. Using specific inhibitors and activators of glycolysis in this protocol, we show how to obtain a systemic and complete view of glycolytic metabolic processes in the cells and provide more accurate and realistic results. To be able to measure multiple glycolytic phenotypes, we provide an easy, sensitive, DNA-based normalization method for polarization assessment of BMDMs. Culturing, activation/polarization and identification of the phenotype and metabolic state of the BMDMs are crucial techniques that can help to investigate many different types of diseases.
In this paper, we polarized the na&#239;ve M0 macrophages to M1-like and M2-like macrophages with LPS and IL4, respectively, and measured a comprehensive set of glycolytic parameters in BMDMs in real-time and longitudinally over time, using extracellular flux analysis and glycolytic activators and inhibitors.

This protocol provides detailed and comprehensive methods for the isolation, culture, polarization, and measurement of the glycolytic metabolic state of live bone marrow-derived macrophages (BMDMs). This paper provides step-by-step instructions with realistic visual illustrations for workflow and glycolytic assessment of BMDMs in real-time.

Macrophages are among the most important antigen-presenting cells. Many subsets of macrophages have been identified with unique metabolic signatures. ...

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 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.

İnsan Viseral Yağ Dokusundan Canlı Adiposit ve Stromal Vasküler Fraksiyonun RNA Analizi ve Makrofaj Fenotiplemesine Uygun İzolasyonu

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