Bu çalışma Victoria Hükümeti&#39;nin OIS Programı tarafından Ulusal Sağlık ve Avustralya Tıbbi Araştırma Konseyi (NHMRC) (526.615 ve 545.906) gelen ve kısmen hibe ile desteklenmiştir. DS NHMRC bir üyesidir.

1. [3H]-kolesterol hazırlanması <ol><li> Çeker ocak içinde bir 1.5 ml mikrofuj tüp (0,5 oyuk başına μCi (19 kBq) Tipik bir deney için gerekli olan) içine [3 H] kolesterol gerekli miktarda dağıtın. </li><li> [3H]-Kolesterol tolüen içinde süspansiyon haline getirildiği takdirde, 1 μCi bir son konsantrasyon% 100 etanol ile N2 gaz ve Pastör pipetiyle (de 37 kBq / ul. Vorteks ve karışım ile tamamen aşağı kurutun. </li></ol> 2. Kaplama Hücreler ve Etiketleme Hücresel kolesterol Insan monosit 4,5, THP-1 insan monosit-makrofajlar 6,7,8, RAW 264.7 murin makrofajlar 5,9,10,11, HeLa hücrelerinde 12, endotel insan umbilikal ven: Bu protokol, aşağıdaki hücre tipleri kullanılarak test edilmiştir trombositler hücreleri (HUVEC), BHK-21 hücreleri 9, insan ve fare fibroblastlar 13,14, HepG2 hücrelerinde insan hepatokarsinoma 15 ve modifiye formunda, &lt;&gt; 16 sup. <ol><li> Hücreleri yeniden süspanse edin ve onları saymak. 0.9 ml tam medyum içinde oyuk başına 6 0.2x10 hücre nihai yoğunlukta 12 oyuklu plakalar haline plakası hücreleri. Hücreler büyümeye devam edecek ve efflux deney zaman iyi başına 0.8x10 6 hücre ulaşacak. Gibi farklılaştırılmış THP-1 hücreleri gibi bölmek değil hücreler için, ekim yoğunluğu başına iyi 0.8x10 6 hücre olmalıdır. Sırasıyla veya 24 de plaka, çift ya da başına hücre sayısının yarısından - 6 kullanıyorsanız. Kuyu sayısı her deneysel koşul için dört nüsha tayini için yeterli olmalıdır. </li><li> THP-1 hücrelerinin farklılaşmasını gerekli ise, 48-72 saat boyunca ortam PMA (nihai konsantrasyon 0.1 ug / ml) ilave edilir. </li><li> [3H] kolesterol içeren mikrofüj tüpe ortam küçük bir alikotu ekleyin. </li><li> Tam kültür ortamı ayrı bir alikotu (100 ul / kuyucuk) içine [3H] kolesterol eklemeden önce ortamı ile iki tüpün yıkanır. </li> &lt;li&gt; hücreleri (çukur başına son hacim 1 ml) ile kuyucuklara [3 H] kolesterol içeren ortam ekleyin. Hücrelerinde hiçbir tedavi etiketlenmesi önce gerekli ise, hücreler [3 H] kolesterol içeren orta kaplama olabilir. </li><li> (37 ° C&#39;de,% 5 CO2) hücre kültürü inkübatöre 48 saat boyunca inkübe hücreleri. </li></ol> 3. Denge Kuluçka <ol><li> 48 saat inkübasyondan sonra, mikroskop altında hücrelerin kontrol edin. Sağlıklı oldukları ve yaklaşık% 80 izdiham olduğundan emin olun. </li><li> Ortam [3 H] kolesterol içeren çıkarın. PBS ile hafifçe hücreleri yıkayın. 3 yıkama kez tekrarlayın. </li><li> Serumsuz ortam hazırlayın. Serum serbest-medya; Gerekirse, LXR agonist (örneğin 1-4 mMol / L &#39;TO-901317 gibi) veya cAMP (sadece fare kökenli hücreler için 0.3 mmol / L) ekleyerek hücrelerini aktive eder. </li><li> Her kuyuya 500 mikrolitre serum serbest Medya ekle. </li><li> (37 ° C&#39;de,% 5 CO2) hücre kültürü inkübatöre 18 saat boyunca inkübe edilir. </li></ol> &lt;p class = &quot;jove_title&quot;&gt; 4. Kolesterol akışı Kuluçka <ol><li> Serum içermeyen aracı madde içinde 18 saatlik inkübasyondan sonra, mikroskop altında hücreleri kontrol. (En az% 80 konfluense) sağlıklı ve birleşik olduğundan emin olun. </li><li> Serum içermeyen aracı madde içinde kolesterol akışı alıcısının çözeltisi hazırlayın. Muhatapları örnekler: Apolipoprotein AI (apoA-I) (son konsantrasyonu 10 mg / ml) Yüksek dansiteli lipoprotein (HDL) (son konsantrasyonu 20 mg / ml) Siklodekstrin (nihai konsantrasyon 200 ug / ml) Plazma (son konsantrasyon% 1-2) </li><li> PBS ile hafifçe hücreleri yıkayın. </li><li> Her bir kuyuya muhatapları ile serumsuz medya 250 ul ekleyin. </li><li> Arka plan efflux (NO alıcı ile serum serbest medya efflux) belirlemek için kuyu bir takım bırakın </li><li> Hücre kültürü inkübatörde 2 saat boyunca hücreleri (37 ° C&#39;de,% 5 CO2) inkübe. Gerekirse efflux inkübasyon süresi 30 dakika ile 8 saat değişebilir. </li></ol> 5.. İşleme Samples <ol><li> 2 saat sonra kuluçka mikroskop altında hücrelerin kontrol edin. </li><li> 1.5 ml mikrofuge&#39;de tüpler içine medya toplayın. Hücresel enkaz kaldırmak için oda sıcaklığında 1-10 dakika 14.000 rpm&#39;de Spin. </li><li> 7 ml sintilasyon şişeye 100 ul medya aktarın. 5 Insta-jel Plus mls (Perkin Elmer) ve vorteks karışımı ekleyin. 4 ° C&#39;de geri kalan örnekleri depolamak </li><li> 30 dakika için dondurucuya plakaları yerleştirin. Her kuyucuğa 500 ul dH 2 O ekleyin. Tüm hücreleri kuyu dibinden kaldırdı emin olmak için mikroskop altında hücrelerin kontrol edin. Hala yapışık ise, 4 ° C de bir gece veya sıyrık kuyularında dH 2 O ile plakalar bırakın ya. </li><li> Tüm hücreler kaldırdı sonra, hücre kümeleri kırmak için aşağı ve yukarı Pipeti. 7 ml sintilasyon vial içine 100 ul aktarın. 5 Insta-jel Plus mls (Perkin Elmer) ve vorteks karışımı ekleyin. 4 ° C&#39;de geri kalan plakaları depolamak </li><li> Sintilasyon sayacı sintilasyon şişelerine yerleştirin. [3 H] için saymak için sayaç yapılandırındpm birimleri. </li></ol> 6. Sonuçları analiz <ol><li> Kolesterol akışı oranı, genellikle kolesterol bir oranda hücrelerinden alıcı taşınır olarak ifade edilir. Aşağıdaki formül kullanılır: </li></ol><img alt="Denklem 1" src="/files/ftp_upload/3810/3810eq1.jpg" /><ol start="2"><li> Belirli bir akış alıcı (boşluk) varlığında veya yokluğunda, akış arasındaki fark olarak hesaplanır. </li></ol><img alt="Denklem 2" src="/files/ftp_upload/3810/3810eq2.jpg" /> 7. Timeline <img alt="Şekil 1" src="/files/ftp_upload/3810/3810timeline.jpg" /> 8. Temsilcisi Sonuçlar Bir kolesterol akışı deneyde bir sonucu bir örneği, Şek. 1. Bu deneyde THP-1 insan monosit oldu farklı kabul eden makrofaj ve kolesterol akışı içine ayırtlanmıştırtest edilmiştir. Hiçbir alıcıları (&quot;boş&quot;) ile orta kolesterol akışı% 0.79 idi ve bu değer bir non-spesifik akış olarak kabul edilmiş ve diğer değerleri çıkarıldı. İnsan ApoA-I (nihai konsantrasyon 30 ug / ml) ile kolesterol akışı% 4.75 olmuştur. Bir başvuru plazma örneği arası deneysel değişkenlik izlemek için, bu deney dahil edildi. Bir referans örnek deney boyunca çok sayıda veri normalizasyon için kullanılır, ama değişkenliği yüksek olup olmadığını test tekrar bu ihtiyatlı bulunabilir. Hastanın ilacı ile muamele edilmiş, önce ve sonra bir hasta plazma (son konsantrasyon% 2) test edilmiştir. Bu hastada ilaç kolesterol akışı desteklemek için plazma kapasitesi üzerinde olumsuz etkilere sahip olduğu sonucuna varılmıştır. Efflux deneyde bir sonucu başka bir örneği şekil gösterilmektedir. 2. Bu deneyde RAW 264.7 makrofaj LXR agonisti TO-901.317 ile birlikte bir gece inkübasyon (son konsantrasyon ile aktive veya devreye alınmadı1 mmol / L) ve insan plazma aynı örnek (% 2) kolesterol akışı test edildi. Bu LXR agonisti ile ABC taşıyıcıları hücresel ifade aktivasyon ekstrasellüler alıcı için kolesterol serbest hücrelerinin kapasitesini artırır sonucuna varılmıştır. <img alt="Şekil 1" src="/files/ftp_upload/3810/3810fig1.jpg" /> Şekil 1.. Çeşitli alıcıları için THP-1 hücrelerinden kolesterol akışı. Kolesterol akışı yüzdesi (yani etiketli kolesterol oranı hücrelerinden belirtilen alıcı taşındı) boş değerler çıkarma sonra gösterilmiştir. Ortalama ± dörderli tayin SD, * p &lt;0.05. <img alt="Şekil 2" src="/files/ftp_upload/3810/3810fig2.jpg" /> Şekil 2. Kolesterol akışı RAW 264.7 insan plazma LXR agonisti ile aktif ya da aktif değil. Yüzde (yani etiketli kolesterol oranı ile kolesterol akışı hücrelerden taşındıbelirtilen alıcı) boş değerler çıkarma sonra gösterilmiştir. Ortalama ± dörderli tayin SD, * p &lt;0.001.

<ol>
	<li>Tabas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+in+health+and+disease.">Cholesterol in health and disease.</a> J. Clin. Invest. 110, 583-590 (2002).</li><li>Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholesterol+feedback:+from+Schoenheimer's+bottle+to+Scap's+MELADL.">Cholesterol feedback: from Schoenheimer's bottle to Scap's MELADL.</a> J. Lipid Res. 50, 15-27 (2009).</li><li>Tabas, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+cellular+cholesterol+accumulation:+basic+concepts+and+physiological+implications.">Consequences of cellular cholesterol accumulation: basic concepts and physiological implications.</a> J. Clin. Invest. 110, 905-911 (2002).</li><li>Murphy, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Density+Lipoprotein+Reduces+the+Human+Monocyte+Inflammatory+Response.">High-Density Lipoprotein Reduces the Human Monocyte Inflammatory Response.</a> Arterioscler. Thromb. Vasc. Biol. 28, 2071-2077 (2008).</li><li>Mujawar, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Immunodeficiency+Virus+Impairs+Reverse+Cholesterol+Transport+from+Macrophages.">Human Immunodeficiency Virus Impairs Reverse Cholesterol Transport from Macrophages.</a> PLoS Biology. 4, 365-36 (2006).</li><li>Patel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+High-Density+Lipoprotein+Increases+Plasma+High-Density+Lipoprotein+Anti-Inflammatory+Properties+and+Cholesterol+Efflux+Capacity+in+Patients+With+Type+2+Diabetes.">Reconstituted High-Density Lipoprotein Increases Plasma High-Density Lipoprotein Anti-Inflammatory Properties and Cholesterol Efflux Capacity in Patients With Type 2 Diabetes.</a> J. Am. Coll. Cardiol. 53, 962-971 (2009).</li><li>Shaw, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infusion+of+reconstituted+high-density+lipoprotein+leads+to+acute+changes+in+human+atherosclerotic+plaque.">Infusion of reconstituted high-density lipoprotein leads to acute changes in human atherosclerotic plaque.</a> Circ. Res. 103, 1084-1091 (2008).</li><li>Kekulawala, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+freezing+on+high-density+lipoprotein+functionality.">Impact of freezing on high-density lipoprotein functionality.</a> Anal. Biochem. 379, 75-77 (2008).</li><li>D'Souza, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure/Function+Relationships+of+Apolipoprotein+A-I+Mimetic+Peptides:+Implications+for+Antiatherogenic+Activities+of+High-Density+Lipoprotein.">Structure/Function Relationships of Apolipoprotein A-I Mimetic Peptides: Implications for Antiatherogenic Activities of High-Density Lipoprotein.</a> Circ. Res. 107, 217-227 (2010).</li><li>Tchoua, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cholesteryl+ester+transfer+protein+overexpression+and+inhibition+on+reverse+cholesterol+transport.">The effect of cholesteryl ester transfer protein overexpression and inhibition on reverse cholesterol transport.</a> Cardiovasc Res. 77, 732-739 (2008).</li><li>Mukhamedova, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+apolipoprotein+A-I-dependent+cholesterol+efflux+elevates+cholesterol+export+from+macrophages+in+vivo.">Enhancing apolipoprotein A-I-dependent cholesterol efflux elevates cholesterol export from macrophages in vivo.</a> J. Lipid Res. 49, 2312-2322 (2008).</li><li>Mukhamedova, N., Fu, Y., Bukrinsky, M., Remaley, A. T., Sviridov, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Different+Regions+of+ATP-Binding+Cassette+Transporter+A1+in+Cholesterol+Efflux.">The Role of Different Regions of ATP-Binding Cassette Transporter A1 in Cholesterol Efflux.</a> Biochemistry. 46, 9388-9398 (2007).</li><li>Sviridov, D., Fidge, N., Beaumier-Gallon, G., Fielding, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+A-I+stimulates+the+transport+of+intracellular+cholesterol+to+cell-surface+cholesterol-rich+domains+(caveolae.">Apolipoprotein A-I stimulates the transport of intracellular cholesterol to cell-surface cholesterol-rich domains (caveolae.</a> Biochem. J. 358, 79-86 (2001).</li><li>Smyth, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+of+harlequin+ichthyosis+delineates+a+key+role+for+abca12+in+lipid+homeostasis.">A mouse model of harlequin ichthyosis delineates a key role for abca12 in lipid homeostasis.</a> PLoS Genet. 4, e1000192-e1000192 (2008).</li><li>Fu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Caveolin-1+Enhances+Cholesterol+Efflux+in+Hepatic+Cells.">Expression of Caveolin-1 Enhances Cholesterol Efflux in Hepatic Cells.</a> J. Biol. Chem. 279, 14140-14146 (2004).</li><li>Calkin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituted+High-Density+Lipoprotein+Attenuates+Platelet+Function+in+Individuals+With+Type+2+Diabetes+Mellitus+by+Promoting+Cholesterol+Efflux.">Reconstituted High-Density Lipoprotein Attenuates Platelet Function in Individuals With Type 2 Diabetes Mellitus by Promoting Cholesterol Efflux.</a> Circulation. 120, 2095-2104 (2009).</li></ol>

Yazarlar bu çalışma ile ilgili hiçbir çatışan çıkarları beyan ederim.

Kolesterol akışı ölçmek için açıklanan yönteme hücreleri bir hücre dışı kolesterol alıcı için kolesterol hareketinin ölçülmesi için tasarlanmıştır. Bu metodoloji anlamada çok önemli hususlar vardır. Etiketleme ve alışım Tarif metodolojide etiketli kolesterol serumu içeren serum ilave edilir. Ayrıntılı olarak incelenmiştir asla rağmen, kolesterol serum lipoprotein içine dahil edilmiştir ve hücreleri tarafından alınmasını kab...

<table border="1"><tbody><tr><td> Reaktifler \ Hücreleri </td><td> HeLa </td><td> THP-1 </td><td> RAW </td><td> HUVEC </td><td> BHK-21 </td><td> HFF </td></tr><tr><td> Komple Medya </td><td colspan="3"> RPMI -% 10 FCS -% 1 L-glütamin -% 0,2 Kalem strep </td><td> M199 -% 5 FCS -% 1 L-glütamin -% 1 Pen Strep -% 2 HEPES (1M) -% 7 endotel hücre büyümesi takviyesi (EKG) </td><td colspan="2"> DMEM -% 10 FCS -% 1 L-glütamin -% 0,2 Kalem strep </td></tr><tr><td> Serum Ücretsiz Media </td><td colspan="3"> RPMI -% 1 L-glütamin -% 0,2 Kalem strep </td><td> M199 -% 1 L-glütamin -% 1 Pen Strep -% 2 HEPES (1M) -% 7 endotel hücre büyümesinith takviyesi (EKG) </td><td colspan="2"> DMEM -% 1 L-glütamin -% 0,2 Kalem strep </td></tr><tr><td> Passage Yöntemi </td><td> 1 x Tripsin </td><td> Süspansiyon </td><td> Kazımak </td><td colspan="3"> 1 x Tripsin </td></tr><tr><td> Passage Oranı </td><td> 03:10 </td><td> 01:03 </td><td></td><td> 01:20 </td><td> 01:03 </td><td> 01:03 </td></tr><tr><td> Hücre Etkinleştirme (opsiyonel) </td><td></td><td colspan="5"> TO-901.317 - LXR-agonist - Final kons. 4 uM </td></tr><tr><td> Diğer Reaktifler </td><td></td><td> forbol miristat-12 13-asetat (PMA) - Hücre farklılaşması - Final kons. 0.1μg/ml </td><td></td><td></td><td></td><td></td></tr></tbody></table>

cholesterol efflux assay

Hücrelerin kolesterol içeriği hücre zarının bozulması, apoptoz ve nekroz 1 bir hücreye sonuçları çok fazla veya çok az kolesterol, çok dar sınırlar içinde tutulmalıdır. Hücreler hücre içi sentezi ve plazma lipoprotein kaynak kolesterol, hem de kaynakları tamamen kolesterol için hücrelerin ihtiyaçlarını karşılamak için yeterli olabilir. Kolesterol sentezi ve alım süreçleri sıkı regüle ve kolesterol eksikliği azdır 2 olarak uygulanır. Aşırı kolesterol daha yaygın sorun 3&#39;tür. Hepatositlerin dışında ve bir dereceye adrenokortikal hücrelere, hücreler kolesterol düşmesine edemiyoruz. Hücreler kendi kolesterol miktarını azaltmak için iki seçeneğiniz var: aynı zamanda toksik kolesterol esterleri ile hücrelerin aşırı olarak kolesterol esterleri, sınırlı kapasiteli bir seçenek haline dönüştürmek kolesterol ve kolesterol akışı, potansiyel olarak sınırsız kapasiteye sahip bir seçenek. Kolesterol akışı bir specBöyle ATP bağlayıcı kaset taşıyıcı proteinler A1 (ABCA1) ve G1 (ABCG1) ve çöpçü reseptör tip B1 gibi hücre içi nakliyeciler, bir dizi ile düzenlenmiştir IFIC süreci. Plazma kolesterol doğal alıcı yüksek dansiteli lipoprotein (HDL) ve apolipoprotein AI olduğunu. Kolesterol akışı assay kültürü hücrelerinden kolesterol akışı oranı ölçmek için tasarlanmıştır. Bu kolesterol akışı ve / veya hücre serbest kolesterol kabul plazma alıcıları kapasitesini korumak için hücre kapasitesi ölçer. Deneyi aşağıdaki adımlardan oluşur. Adım 1: serum içeren orta etiketli kolesterol ekleme ve 24-48 saat hücreleri ile kuluçkaya hücresel kolesterol etiketleme. Bu adımda kolesterol hücrelerinin yüklenmesi ile kombine edilebilir. Adım 2: bütün hücre içi kolesterol havuzlarına etiketli kolesterol dengelenmeye serum içermeyen aracı madde içinde hücrelerinin inkübasyon. Bu aşamada hücresel kanal aktivasyonu ile kombine edilebilirolesterol taşımacıları. Adım 3: ekstraselüler alıcı ve hücreler arası alıcı için etiketli kolesterol hareketinin kantitatif sahip hücrelerin inkübasyon. Kolesterol öncüleri yeni sentezlenen kolesterol etiketlemek için kullanılması durumunda, bir dördüncü aşama, kolesterol saflaştırma, gerekli olabilir. Deneyi aşağıdaki bilgi sağlar: (i) belirli bir işlem (a mutasyonu, bir itici, aşağı, bir aşırı ekspresyonu ya da tedavi) efflux kolesterol hücre kapasitesi etkiler ve (ii) nasıl plazma alıcısının kapasitesi kolesterol kabul nasıl Bir hastalık veya bir tedavi tarafından etkilenir. Bu yöntem, çoğu zaman kardiyovasküler araştırmalar, metabolik ve nörodejeneratif hastalıklar, bulaşıcı hastalıklar ve üreme bağlamında kullanılır.

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Cholesterol Efflux Assay | Medicine | Video

Cholesterol Efflux Assay

Kolesterol tahlilini kültürü hücrelerinden kolesterol akışı hızı ve hücreler serbest kolesterol kabul plazma alıcısının kapasitesi ölçmek için tasarlanmıştır. Tahlil içi havuzu ve ekstrasellüler alıcı için kolesterol serbest bırakılması arasında kolesterol kolesterol, denge ile hücrelerin etiketlenmesi oluşur.

Kolesterol akışı Testi

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

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29.5K Görüntüleme.  Baker IDI Heart and Diabetes Institute. Kolesterol tahlilini kültürü hücrelerinden kolesterol akışı hızı ve hücreler serbest kolesterol kabul plazma alıcısının kapasitesi ölçmek için tasarlanmıştır. Tahlil içi havuzu ve ekstrasellüler alıcı için kolesterol serbest bırakılması arasında kolesterol kolesterol, denge ile hücrelerin etiketlenmesi oluşur.

Video: Cholesterol Efflux Assay

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Deneysel metastaz Testi

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

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In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

Vivo Köpek Kas Fonksiyon Testi

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

Anjiyogenez Çalışması Kornea Cep Testi Fare Modeli

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

Spheroid Assay to Measure TGF-&amp;#946;-induced Invasion

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

TGF-β-kaynaklı Invasion Tedbir Sfero Testi

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

An<em&gt; In vivo</emKan Gemi Geçirgenlik Test&gt; Test

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

<em&gt; Ovo</emSarkoma için bir Xenograftmodel olarak&gt; CAM-testi

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the pathophysiological role of neuroplasticity in GI disorders remains unknown. Novel experimental models which allow simulation and modulation of GI neuroplasticity may enable enhanced appreciation of the contribution of neuroplasticity in particular GI diseases such as pancreatic cancer (PCa) and chronic pancreatitis (CP). Here, we present a protocol for simulation of pancreatic neuroplasticity under in vitro conditions using newborn rat dorsal root ganglia (DRG) and myenteric plexus (MP) neurons. This dual-neuron approach not only permits monitoring of both organ-intrinsic and -extrinsic neuroplasticity, but also represents a valuable tool to assess neuronal and glial morphology and electrophysiology. Moreover, it allows functional modulation of supplied microenvironmental contents for studying their impact on neuroplasticity. Once established, the present neuroplasticity assay bears the potential of being applicable to the study of neuroplasticity in any GI organ.

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

Pankreas Nöroplastisite benzetme:<em&gt; In Vitro</em&gt; Dual-nöron Plastisitesi Deneyi

Neuroplasticity is an inherent feature of the enteric nervous system and gastrointestinal (GI) innervation under pathological conditions. However, the ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Üç boyutlu (3D) Tümör Sfero Yayılması deneyindeki

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Differential Effects of Lipid-lowering Drugs in Modulating Morphology of Cholesterol Particles

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to moderate level of increase in high-density lipoprotein (HDL) cholesterol in plasma. However, a possible role of these drugs in altering morphology and distribution of cholesterol particles is poorly understood. Here, we describe the in vitro evaluation of lipid-lowering drug effects in modulating morphological features of cholesterol particles using the plaque array method in combination with imaging flow cytometry. Image analyses of the cholesterol particles indicated that lovastatin, simvastatin, ezetimibe, and atorvastatin induce the formation of both globular and linear strand-shaped particles, whereas niacin, fibrates, fluvastatin, and rosuvastatin induce the formation of only globular-shaped particles. Next, purified very low-density lipoprotein (VLDL) and LDL particles incubated with these drugs showed changes in the morphology and image texture of cholesterol particles subpopulations. Furthermore, screening of 50 serum samples revealed the presence of a higher level of linear shaped HDL cholesterol particles in subjects with dyslipidemia (mean of 18.3%) compared to the age-matched normal (mean of 11.1%) samples. We also observed considerable variations in lipid-lowering drug effects on reducing linear shaped LDL and HDL cholesterol particles formation in serum samples. These findings indicate that lipid-lowering drugs, in addition to their cell-mediated hypolipidemic effects, may directly modulate morphology of cholesterol particles by a non-enzymatic mechanism of action. The outcomes of these results have potential to inform diagnosis of atherosclerosis and predict optimal lipid-lowering therapy.

The objective of this study was to evaluate in vitro lipid-lowering drug effects in modulating the morphology of cholesterol particles. Comparison of lipid-lowering drugs revealed variations in their effect in modulating the morphological features of cholesterol particles.

Kolesterol parçacıkları morfolojisi oransal olarak Lipid düşürücü ilaçlar farklı etkileri

Treatment of dyslipidemia patients with lipid-lowering drugs leads to a significant reduction in low-density lipoproteins (LDL) level and a low to ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.