Name: saturated fatty acids induce ceramide-associated macrophage cell death
Uploaded: 2017-10-31T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death at JoVE.com

Bu eser kısmen Louisville Üniversitesi başlangıç fonlar tarafından desteklendi ve Ulusal Kanser Enstitüsü (Bethesda, MD) R01CA177679, R01CA180986 verir.

protokol kurumsal hayvan bakım ve kullanım Komitesi (IACUC), Louisville Üniversitesi tarafından kabul edildi.
 1. fare kemik iliği türetilmiş makrofajlar (BMDMs) <ol><li>Euthanize CO 2 kullanarak bir 6 - için 8-hafta-yaşlı, sağlıklı vahşi tip fare. Bir köpük tahta aşağı pin ve sırılsıklam olana % 70 etanol ile sprey. Tibia/uyluk kemikleri forseps ve makas ile kaldırın. Onları 5 mL 1 x fosfat tamponlu tuz çözeltisi (PBS) ile petri kabına koyardım. Steril koşullar altında tüm yordamları gerçekleştirmek.</li>
	<li>Her iki ucunda da bir makas ile tibia/uyluk kemiği steril doku kültürü mahallede kesmiş. 15 mL tüp içine kemik PBS + % 2 fetal sığır serum (FBS) ile 25 G iğne ile flush.</li>
	<li>Kemik iliği hücreleri vasıl 500 x g, 4 ° C'de 5 dakika süreyle santrifüj kapasitesi</li>
	<li>Hücre Pelet 1 mL kırmızı kan hücre lizis arabelleği kırmızı kan hücreleri (RBC) koşullar için daha az 1 dk. Dilute 9 mL 1 x PBS ile hemen resuspend. Santrifüj tekrar adım 1.3 olduğu gibi. Supernatants dikkatle boşaltmak.</li>
	<li>10 mL 1 x PBS içinde hücre Pelet resuspend, hücre süspansiyon hücre enkaz/kümeleri kaldırmak için başka bir 15 mL tüp içine steril 40 mikron naylon mesh aracılığıyla filtre. Santrifüj tekrar 1.3 olduğu gibi. Supernatants dikkatle boşaltmak.</li>
	<li>Hücre Pelet 15 mL Roswell Park Memorial Enstitüsü orta (RPMI) % 5 ile 1640 FBS ve 10 μg/mL gentamisin, yüzen dışarı 100-mm doku kültürü çanak 37 o C İnkübatör, hücrelerde alın 60 dk. yavaşça girdap için çanak, tabak resuspend hücreleri ve bunları kabul.</li>
	<li>6 x 10 6 hücreler bir 100-mm tabak içinde 10 ng/mL rekombinant fare makrofaj ile medya (%5 FBS ve 10 μg/mL gentamisin, % 30 ' L929 şartına media 10 ile karışık RPMI 1640) ayırt 12 mL makrofaj ile plaka 2 gün için koloni uyarıcı faktör (M-CSF).</li>
	<li>% 5 CO 2 37 ° C kuluçka 2 gün boyunca kültür sonra her yemeğin medya 10 ng/mL M-CSF ile ayırt 6 mL taze makrofaj ile beslemek.</li>
	<li>5 günde 8 mL eski medya hücreleri rahatsız etmeden alıp 10 mL taze makrofaj medya 10 ng/mL M-CSF ile ayırt ekleyin.</li>
	<li>7 günde bir hücre lifter kullanarak kemik iliği türevi makrofajlar (BMDMs) hasat. 
	Not: Ekli makrofajlar da 5 mL 1 mM EDTA 1 x PBS için 5-10 dk içinde ile float hücreleri kaldırma ve iki kez 5 mL 1 x PBS ile tabak yıkama sonra ayrışmış. Adım 1.3 olduğu gibi hücreleri santrifüj kapasitesi., onları taze makrofaj medya ayırt resuspend ve bir hemasitometre ile Sayın.
	<ol><li>Onayla BMDMs &#39; fenotip Akış Sitometresi tarafından açıklanan 5. Türetilmiş fare makrofajlar aşağıdaki çalışmalar için belirli bir konsantrasyon hazırlayın.</li>
	</ol></li></ol> 2. BSA-yağ asit eşlenik hazırlık <ol><li>hazırla 2 mM BSA steril PBS. Örneğin, tartmak 6,65 g BSA, çözülmeye, 30 mL 1 x PBS eklemek sonra eklemek daha fazla PBS 50 mL kadar bileşenleri 2 mM BSA 1 x PBS yapmak için ölçekleme.</li>
	<li>Hazırlama 5 mM bireysel sodyum tuzu 2 mM BSA yağ asitlerinin. 37 ° C veya daha yüksek, gerekirse ısı ve net bir çözüm elde edilir kadar 2 mM BSA karışımı ve yağ asitlerinin sodyum tuzu solüsyon içeren temizleyicide. 
	Not: doymamış yağ asitleri için karşılaştırıldığında, doymuş yağ asitleri daha yüksek bir sıcaklık ve sonication zamana eriterek için gerek.</li>
	<li>Filtre bir 0,22 ile yağ-asit-BSA çözüme mikron filtre, aliquot 1,5 mL steril microcentrifuge içine borular, onları kısa süreli kullanım için 4 ° C'de veya uzun süreli depolama için-20 ° C'de ve saklayabilirsiniz. Hiç yağış depolama buzdolabında 4 ° C'de sonra dikkat edilmelidir.</li>
</ol> 3. Doymuş yağ asitleri neden hücre ölüm, fare kemik iliği türetilmiş makrofajlar <ol><li>BMDMs 24-iyi doku kültürü plaka 10 6 /mL medya ayırt makrofaj olarak x 0.4 ile plaka.</li>
	<li>0.4 mM palmitik asit ve Stearik asit, 37 ° C kuluçka %5 CO 2 ile 16-24 h için hücrelerle tedavi. Negatif kontrol BSA kullanın.</li>
	<li>Tedaviden sonra hücre lifter hücrelerle hasat ve spin 500 x g, 4 ° C'de 5 dakika süreyle, hücreleri aşağı</li>
	<li>Annexin V-Alexa 488 ve 7-aminoactinomycin D (7-AAD) 100 hücrelerle leke μL V bağlama arabellek 15 dakika oda sıcaklığında annexin.</li>
	<li>Annexin-bağlama arabellek 200 µL örnekle sulandırmak, karışımı yavaşça, o zaman buz üzerinde örnekleri kuluçka süresinden sonra devam.</li>
	<li>Lekeli hücreleri en kısa zamanda Akış Sitometresi 5 tarafından analiz. V-Alexa 488 annexin FITC kanalda algılandığında ve 7-AAD PerCP/PerCP-Cy5.5 kanalda algılandı.</li>
</ol> 4. Hücre içi Ceramide boyama <ol><li>yağ asidi tedaviden sonra adım 3.3 olduğu gibi hücre hasat. % 4 paraformaldehyde 30 dakika süreyle hücreleri tamir</li>
	<li>Örnek 1 mL 1 x PBS ile yıkayın, aşağı spin olarak 3.3 içinde adım ve süpernatant dikkatle boşaltmak.</li>
	<li>100 μL 1 x permeabilization tampon 30 dakika süreyle anti-ceramide birincil antikor (1: 200 seyreltme) hücrelerle leke</li>
	<li>Yıkama ve spin aşağı hücreleri tekrar olduğu gibi adım 4.2.</li>
	<li>100 μL 1 x permeabilization tampon 30 dakika süreyle Anti-fare IgM ikincil antikor (1:500 seyreltme) hücrelerle leke</li>
	<li>Yıkama ve spin adım 4.2 olduğu gibi yine hücreleri aşağı. 250 μL 1 x PBS ekleyin ve lekeli hücreleri Akış Sitometresi 5 tarafından analiz. 
	Not: ekipmanları ve Kimyasalları tablosu ayrıntılı reaktif listesinde bakın.</li>
</ol>

<ol>
	<li>Martins de Lima, T., Cury-Boaventura, M. F., Giannocco, G., Nunes, M. T., Curi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+toxicity+of+fatty+acids+on+a+macrophage+cell+line+(J774).">Comparative toxicity of fatty acids on a macrophage cell line (J774).</a> Clin Sci (Lond). 111 (5), 307-317 (2006).</li><li>Simard, J. R., Zunszain, P. A., Hamilton, J. A., Curry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+high+and+low+affinity+fatty+acid+binding+sites+on+human+serum+albumin+revealed+by+NMR+drug-competition+analysis.">Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis.</a> J Mol Biol. 361 (2), 336-351 (2006).</li><li>Penn, A. H., Dubick, M. A., Torres Filho, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+Acid+Saturation+of+Albumin+Used+in+Resuscitation+Fluids+Modulates+Cell+Damage+in+Shock:+In+Vitro+Results+Using+a+Novel+Technique+to+Measure+Fatty+Acid+Binding+Capacity.">Fatty Acid Saturation of Albumin Used in Resuscitation Fluids Modulates Cell Damage in Shock: In Vitro Results Using a Novel Technique to Measure Fatty Acid Binding Capacity.</a> Shock. , (2017).</li><li>Vusse, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Albumin+as+fatty+acid+transporter.">Albumin as fatty acid transporter.</a> Drug Metab Pharmacokinet. 24 (4), 300-307 (2009).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+Fatty+Acid+Binding+Protein+Promotes+Saturated+Fatty+Acid-Induced+Macrophage+Cell+Death+through+Enhancing+Ceramide+Production.">Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-Induced Macrophage Cell Death through Enhancing Ceramide Production.</a> J Immunol. 198 (2), 798-807 (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+Fatty+Acid+binding+protein+promotes+skin+inflammation+induced+by+high-fat+diet.">Epidermal Fatty Acid binding protein promotes skin inflammation induced by high-fat diet.</a> Immunity. 42 (5), 953-964 (2015).</li><li>Wen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid-induced+NLRP3-ASC+inflammasome+activation+interferes+with+insulin+signaling.">Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling.</a> Nat Immunol. 12 (5), 408-415 (2011).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid-binding+protein+E-FABP+restricts+tumor+growth+by+promoting+IFN-beta+responses+in+tumor-associated+macrophages.">Fatty acid-binding protein E-FABP restricts tumor growth by promoting IFN-beta responses in tumor-associated macrophages.</a> Cancer Res. 74 (11), 2986-2998 (2014).</li><li>Ulloth, J. E., Casiano, C. A., De Leon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+and+stearic+fatty+acids+induce+caspase-dependent+and+-independent+cell+death+in+nerve+growth+factor+differentiated+PC12+cells.">Palmitic and stearic fatty acids induce caspase-dependent and -independent cell death in nerve growth factor differentiated PC12 cells.</a> J Neurochem. 84 (4), 655-668 (2003).</li><li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow-Derived+Macrophages+(BMM):+Isolation+and+Applications.">Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.</a> CSH Protoc. , (2008).</li><li>Dansen, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+binding+of+very-long-chain+fatty+acyl-CoA+esters+to+the+peroxisomal+non-specific+lipid-transfer+protein+(sterol+carrier+protein-2).">High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2).</a> Biochem J. 339 (Pt 1), 193-199 (1999).</li><li>Ulloth, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+methyl-beta-cyclodextrin+toxicity+in+NGF-differentiated+PC12+cell+death.">Characterization of methyl-beta-cyclodextrin toxicity in NGF-differentiated PC12 cell death.</a> Neurotoxicology. 28 (3), 613-621 (2007).</li></ol>

Yazarlar hiçbir maddi çıkar çatışmaları bildirin.

Yağ asidi çözüm uygun hazırlanması yağ asitlerinin biyolojik işlev eğitim için kritik öneme sahiptir. Yağ asidi nötralize sulu çözüm onun çözünürlük artar. Ancak, hala su veya PBS düşük çözünürlük, yağ asitlerinin, özellikle doymuş yağ asitleri, sodyum tuzları şunlardır biz gözlenen. Bir yöntem yağ asidi1dağıtılması yardımcı olmak için % 95-100 etanol kullanmaktır. Bu yöntemi kullanarak, hücrelere daha yüksek toksisite daha az toksik doymamış ya?...

Yağ asitleri enerji metabolizması ve farklı hücre membran fosfolipitler sentezi kritik bir rol oynamaktadır. Yağ asitleri sulu düşük bir çözünürlük var. Yağ asidi uygun hazırlanması biyolojik fonksiyonları memeli hücrelerinde yağ asitlerinin çalışmak için kritik önem taşıyor. Yağ asitleri etanol ile hazırlanır birçok yağ asitleri bile nispeten düşük konsantrasyonlarda1hücre zarının toksik sabun (deterjan) etkileri göstermek. Bir doğal, büyük taşıyıcı serum serbest yağ asitleri için serum albümin yağ asidi teslim vitro yağ asidi işlevi deneyleri için2,3,4için iyi bir taşıyıcı olarak kabul edilir. Birçok araştırma kağıtları yağ asitleri kullanarak yayımlanmış olsa bile ancak, yağ asidi ve serum albümin eşlenik hazırlanması ayrıntılarını genellikle kullanılabilir değil.
Makrofajlar epidermal yağ asidi bağlayıcı protein ve yağ yağ asidi bağlayıcı protein5,6,7,8son derece hızlı. Onlar aktif alımı doymuş ve doymamış yağ asitleri bağışıklık işlevleri düzenleyen. Makrofajlar ve diğer hücrelerin yağ asitlerinin etkisi çalışma için uygulanan1,7,9yağ asidi hazırlık farklı yöntemleri vardı. Yağ asitleri makrofaj fonksiyonları üzerine etkisini araştırmak için uygun şekilde hazırlanmış yağ asidi/serum albümin conjugates kullanarak biyolojik anlamlı veri almak üzere kritik önem taşımaktadır. Yağ asitleri makrofaj fonksiyonları üzerine etkisi üzerinde çalışmalar temel bilgi ve yağ asidi metabolizmasının hastalıklarda makrofaj söz konusu ile ilgili potansiyel tedavi hedefleri sağlar.

<table><tbody><tr><td>CPX Ultrasonic Bath&amp;nbsp;</td><td>Bransonic</td><td>Model 2800</td><td></td></tr><tr><td>Sodium palmitate (PA)</td><td>Nu-Chek Prep, Inc.</td><td>S-1109</td><td>M.W. 278</td></tr><tr><td>Sodium stearate (SA)</td><td>Nu-Chek Prep, Inc.</td><td>S-1111</td><td>M.W. 306</td></tr><tr><td>Bovine serum albumin (BSA), fatty acid-free</td><td>Fisher Scientific</td><td>9048-46-8</td><td></td></tr><tr><td>Mouse macrophage colony stimulating factor (mM-CSF)&amp;nbsp;</td><td>Cell Signaling Technology,&amp;nbsp; Inc.</td><td>5228</td><td></td></tr><tr><td>RPMI 1640</td><td>VWR International</td><td>71002-878</td><td></td></tr><tr><td>Annexin V, Alexa Fluor 488 conjugate</td><td>Fisher Scientific</td><td>A13201</td><td></td></tr><tr><td>7-AAD</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>Monoclonal anti-ceramide antibody (mouse IgM)</td><td>Sigma</td><td>C8104-50TST</td><td>Clone: MID 15B4</td></tr><tr><td>Goat Anti-Mouse IgM Antibody, &amp;micro; chain, FITC conjugate</td><td>Sigma</td><td>AP128F</td><td></td></tr><tr><td>Fixation buffer</td><td>Biolegend</td><td>420801</td><td></td></tr><tr><td>Permeabilization buffer</td><td>Ebioscience</td><td>4307693</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer&amp;nbsp;</td><td>Sigma</td><td>11814389001</td><td></td></tr><tr><td>Annexin V Binding Buffer</td><td>BD Biosciences</td><td>556454</td><td></td></tr><tr><td>L929 cells</td><td>ATCC</td><td>CCL-1</td><td></td></tr><tr><td>Corning&amp;nbsp;Cell Lifter&amp;nbsp;</td><td>Fisher Scientific</td><td>07-200-364</td><td></td></tr><tr><td>Note: M.W. is for molecular weight.</td><td></td><td></td><td></td></tr></tbody></table>

saturated fatty acids induce ceramide-associated macrophage cell death

Makrofajlar epidermal yağ asidi bağlayıcı protein ve yağ yağ asidi bağlayıcı protein son derece hızlı. Onlar aktif alımı doymuş ve doymamış yağ asitleri, bağışıklık fonksiyonları düzenlenmesinde önemli bir rol oynayabilir. Çok sayıda çalışmalar çeşitli yağ asitleri, doymuş olduğunu göstermiştir veya doymamış, Mayıs hücre büyümesi ve fonksiyonu farklı etkileri sahip. Ancak, yağ asidi hazırlık için kullanılan yaklaşımlar değişir, hangi fizyolojik olmayan sonuçlara neden olabilir. Serum albümin, memeli periferik kan, yağ asitleri için doğal bir taşıyıcı yağ asitlerinin sodyum tuzu ile karmaşık eşlenik şekillendirme için yağ asidi işlevinde böylece yağ asidi sabun toksisite en aza indirerek memeli hücreleri, eğitim için önerilir. Bu nedenle, basit, nispeten hızlı Isıtma ve yöntem sonicating geliştirilen ve burada BSA-yağ asit eşlenik oluşumu için sunulan. Doymuş yağ asitleri, özellikle Stearik asitler şiddetli hücre ölümü fare kemik iliği türetilmiş makrofajlar içinde ikna etmek için bir iletişim kuralı'nı açıklar. Biz daha fazla doymuş yağ asidi indüklenen hücre ölümü olumlu birikmiş hücresel ceramide düzeyleri ile ilişkili olduğunu gösterir. Bu yöntem yağ asidi diğer memeli hücreleri üzerinde etkisini çalışmaları için genişletilebilir.

Obezite Serumda serbest yağ asitleri konsantrasyonları artar. Profesyonel fagositler makrofajlar aktif olarak ana bilgisayar homeostazı korumak için yağ asitleri götür. Bu işlemler sırasında aşırı yüklenmiş lipidler makrofaj hücre ölümüne neden olabilir. Bu amaçla, biz BMDMs vitro obez düzeyde diyet yağ asitleri ve akış sitometrik boyama kullanarak ölçülen makrofaj hücre ölümü ile kültürlü. Doymuş yağ asitleri, BSA denetimine göre özellikle S...

Watch this Scientific Journal Video about Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death at JoVE.com

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Kemik iliği hücrelerinden fare birincil makrofajlar türetmek için bir düz ileri yöntem göstermek ve BSA-yağ asidi hazırlamak için basit bir yöntem conjugates. Sonra doymuş yağ asitleri makrofaj hücre ölümüne neden olabilir ve bu tür hücre ölümü olumlu ceramide düzeyleri hücresel birikimi ile ilişkilidir gösterir.

Doymuş yağ asitleri Ceramide ilişkili makrofaj hücre ölümüne neden

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

saturated-fatty-acids-induce-ceramide-associated-macrophage-cell-death

Research

JoVE Journal

Immunology and Infection

9.2K Görüntüleme.  University of Louisville. Kemik iliği hücrelerinden fare birincil makrofajlar türetmek için bir düz ileri yöntem göstermek ve BSA-yağ asidi hazırlamak için basit bir yöntem conjugates. Sonra doymuş yağ asitleri makrofaj hücre ölümüne neden olabilir ve bu tür hücre ölümü olumlu ceramide düzeyleri hücresel birikimi ile ilişkilidir gösterir.

Video: Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

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

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

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. ...

İmmünoloji ve Enfeksiyon

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

Manyetik Aktif Hücre Ayırma (LIMACS) kullanarak Lipid veziküllü aracılı Afinite Kromatografisi: Protein-lipit Etkileşim Analiz Roman Yöntemi

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Biyoloji

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasome harekete geçirmek ve Pyroptotic hücre ölümü fare kemik iliği türevi makrofajlar içinde tespiti

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Makrofajlar tarafından apoptotik thymocyte engulfment deneysel Analizi

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

Farklılaşmış HepaRG hücrelerinde veziküler steatozu indükleyen ve karakterize etme

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Tıp

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Hücre içi Komşu maruz kalan hücreler, apoptotik hücre ölümü ile etkileşim yoluyla canlı hücreler İndüklenen sinyal olaylarının tanımlanması

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Lipid Damlacık Boyutu ve Füzyonunun Oil Red O ve BODIPY Kullanılarak Canlı Hücre Görüntülemesi

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Yazar Spotlight: Sığır Hepatik Hücrelerinde Lipid Damlacık Boyutunun ve Füzyonunun Değerlendirilmesi  

The present protocol describes how to use oil red O to dye lipid droplets (LDs), calculate the size and number of LDs in a fatty acid-induced fatty hepatocyte model, and use BODIPY 493/503 to observe the process of small LDs fusing into large LDs by live cell imaging.

Sığır Hepatik Hücrelerinde Lipid Damlacık Büyüklüğü ve Füzyonunun Değerlendirilmesi

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...

THP-1 Hücrelerinde LPS/ATP ile Tetiklenen Hücre Ölüm Mekanizmalarının Analizi

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show&#160;that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

Yazar Spotlight: LPS / ATP'ye THP-1 Makrofaj Yanıtı - Pipoptoz, Apoptoz ve Nekrotoz Spektrumunun Ortaya Çıkarılması

This protocol is based on LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. We use flow cytometry to analyze Annexin V and 7-AAD double staining to detect cell death, using the whole cell and employing scanning electron microscopy to observe the cell membrane morphology.

PMA ile Farklılaşmış THP-1 Makrofajlarının LPS ve ATP ile İndüklenen Ölümü ve Validasyonu

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced ...

Doymuş yağ asitleri Ceramide ilişkili makrofaj hücre ölümüne neden

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Manyetik Aktif Hücre Ayırma (LIMACS) kullanarak Lipid veziküllü aracılı Afinite Kromatografisi: Protein-lipit Etkileşim Analiz Roman Yöntemi

Inflammasome harekete geçirmek ve Pyroptotic hücre ölümü fare kemik iliği türevi makrofajlar içinde tespiti

Makrofajlar tarafından apoptotik thymocyte engulfment deneysel Analizi

Farklılaşmış HepaRG hücrelerinde veziküler steatozu indükleyen ve karakterize etme

Hücre içi Komşu maruz kalan hücreler, apoptotik hücre ölümü ile etkileşim yoluyla canlı hücreler İndüklenen sinyal olaylarının tanımlanması

Sığır Hepatik Hücrelerinde Lipid Damlacık Büyüklüğü ve Füzyonunun Değerlendirilmesi

Sığır Hepatik Hücrelerinde Lipid Damlacık Büyüklüğü ve Füzyonunun Değerlendirilmesi

Sığır Hepatik Hücrelerinde Lipid Damlacık Büyüklüğü ve Füzyonunun Değerlendirilmesi

PMA ile Farklılaşmış THP-1 Makrofajlarının LPS ve ATP ile İndüklenen Ölümü ve Validasyonu