1. CoCl 2 çözüm indüklediği hipoksi Kobalt (II) Klorür hekzahidrat ve (CoCl 2 • 6H 2 O, MW = 237.9), hipoksi-indüklenebilir faktör-1.3 8 indükleyen kimyasal bir madde. Bu ürün, açık, kırmızı bir çözüm verimli, su (100 mg / ml) çözünür. <ol><li> 25mm bir stok solüsyonu steril gg su hazırlayın, (hemen önce kullanıma hazırlamak) </li><li> Hipoksi ikna etmek için normal bir hücre kültürü medya 100μM son konsantrasyonda CoCl 2 kullanın . </li><li> Hücrelere CoCl 2 içeren medya ekleyin ve kültürlerin geleneksel bir inkübatör 24 saat inkübe (37 ° C,% 5 C0 2). </li></ol> Yukarıdaki konsantrasyonu hücre hatları için test ettik ama her hücre hattı, ilaca bağlı toksisite sınırı ve tahlil optimize etmek amacıyla çeşitli inkübasyon sürelerinde yanı sıra çeşitli konsantrasyonlarda (doza bağımlı bir eğri kurmak için) test edilmelidir çalışır. 2. Modüler İnkübatör Odası indüklediği hipoksi <ol><li> En az iki özdeş (ikiz kültür) hücre kültürleri hazırlayın. Hücre kültürleri, şişeler, tabak ya da kültür yemekleri olabilir. Odanın açmak için, ilk oda (hipoksik gaz tasfiye enjeksiyon / için kullanılan tüpler) ve yavaşça, çelik halka kelepçe açık bağlı borular üzerinde bulunan iki beyaz plastik kelepçeleri açın. </li><li> Kelepçe bırakın. Kapak ve tepsiler artık kaldırılabilir. </li><li> Hipoksik odasında hücre kültürü yerleştirin. Ayrıca odasında kültürlerinin yeterli nemlendirme sağlamak için steril su içeren bir Petri kabı yerleştirin. </li><li> Normoxia &quot;ikiz&quot; hücre kültürü kontrolü yerleştirin. </li><li> Tepsiler güvenli ve hareket etmiyor ve sonra kapağı odasına yakın emin olun. Doğru odasının Hermetik kapanmasını sağlamak ve bunu kapatmak için çelik halka kelepçe yerleştirin. </li><li> Hipoksi oluşturmak için,% 1 O 2 gaz karışımı içeren bir &quot;hipoksi tankı&quot; tüp takın. Tankına bağlı bir debimetre varsa odasına doğrudan gaz tankı-debimetre odacıklı () bağlı olacaktır. Biz regülatör kurulmuş bir debimetre kullanın. </li><li> Bunu yapmak için 7-10 dakika dakika başına 20 litre akış hızında gaz tankı açarak odasına floş odasında ve medya tüm oksijen mevcut, eğer en kaldırmak için önemlidir; sonra hızlı bir şekilde kapatmak- gaz akışı ve odanın her iki beyaz kelepçeler kapatarak tamamen kapatın. </li><li> Zaman istenilen süre için geleneksel bir inkübatör odasına dönün. 2 saat sonra tekrar aynı hizada büyük kültürleri kullanıyorsanız, 1 için de gaz kültürlerde medya izin verir. Yukarıdaki adımları yapıcı öneriler &#39;(Billups-Rothenberg, Inc.) Dayanmaktadır. Gaz tankeri, her zaman için güvenli olduğundan emin olun. </li></ol> Notlar: <ul><li> 1-3hours sonra yeniden gaz odasına kez tavsiye medya bulunan O 2 ortadan kaldırmak için. </li><li> 48hours daha uzun inkübasyon süresi için de yeniden gaz odasına tavsiye edilir. </li><li> O 2 hücre / odasında yer alan ölçümleri sağlayan oksijen sensörleri (elektrotlar / manometre), oda / hücre içi O 2 içerir) daha hassas ölçüm sağlayacaktır . </li><li> Bir zaman eğrisi yapmak için çeşitli süre kültür hücrelerinin belli hücreleri HIF-1α uygun zaman ifade belirlemenize yardımcı olacaktır. </li></ul> 3. Hipoksi Değerlendirilmesi <ol><li> Western blot HIF-1 α algılama. <ol><li> Yeniden açın odası olarak bölüm 2.2 &#39;de açıklanan ve hemen buz üzerinde kültürler yerleştirin. </li><li> Plakalar% 5 SDS çözümü ile hipoksi arıtılmış ve arıtılmamış hücreler Lyse sonra, tüpler sonikasyon, yavaşlatmayı hücre artıkları hücre lyzate transferi ve süpernatantı toplamak. </li><li> Tedbir protein konsantrasyonu BCA kiti kullanılarak, 5 dakika boyunca 95 yükleme tamponu ve ısıtma ° C ekleyerek örnek hazırlayın. </li><li> % 8 SDS-poliakrilamid jel ve Selülozik membranlara transfer Electrophorese proteinler. </li><li> 1 saat oda sıcaklığında veya çalkalayıcı üzerinde 4 ° C gecede% 5 yağsız kuru süt içeren PBS membran ve% 0.1 Tween 20 engelleyin. </li><li> 4 aynı tampon anti-HIF-1α birincil monoklonal antikor (1 / 600) bir gecede immüno ° C. </li><li> PBS PBS içinde HRP-ikincil antikor (1 / 2000) +% 0.1 Tween 20 45 dakika oda sıcaklığında, 5dakika her zaman ve inkübe% 0.1 Tween 20 içeren 3 kere yıkayın. </li><li> PBS, 5dakika / saat oda sıcaklığında% 0.1 Tween 20 içeren 3 kere yıkayın. </li><li> Protein bandı göstermek için Pierce ECL kiti ve X-ray film kullanın. </li></ol></li><li> Hipoksi Duyarlı Elemanlar (HRE). HIF-1α Hipoksi Düzenlenmesi Element (HRE) olarak adlandırılan bir dizi bağlanarak çok sayıda genler (yani VEGF, EPO) düzenler. HRE ilişkili bir marker gen I9 HIF aktivite tespit etmek mümkündür. Bu yöntemi kullanmak için, önce hücrelerin adapte transfeksiyon yöntemi kullanılarak HRE lusiferaz plazmid hücreleri transfect gerekir. Örneğin biz Lipofectamine 2000 kullanılır. Hücreler hipoksi ve normoxia inkübe önce, en az 4 saat transfekte olması gerekiyor. Bu sefer, bu ilk adım, hipoksi etkilenmez, böylece DNA&#39;nın hücrelere girmek için izin verir. Lusiferaz sinyal Lusiferaz Assay Kit kullanılarak tespit edilmiştir. <ol><li> Hipoksi, modüler odasında HRE-transfekte hücreler inkübe veya CoCl 2 ile inkübe HRE transfekte hücreleri normoxia kontrolü içerir. </li><li> Istenilen bir kuluçka döneminden sonra, hücre büyümesi orta çıkarın. </li><li> PBS içinde hücreler durulayın, PBS çıkarın. </li><li> Kültür çanak içine 400μl 1X reaktifi Uygulama ve ekli hücreleri kazıyın, sonra onları bir mikro tüp transferi. </li><li> Kısa süreli santrifüj Pelet enkaz. </li><li> Mix 20μl hücre Lusiferaz Testi Reaktif 100μl supernatant lizatları ve lüminesans lüminometre kullanarak ölçmek. </li></ol></li></ol> 4. Temsilcisi Sonuçlar: K562 hücrelerinin (insan lösemi hücre hattı) normoxia kıyasla düşük oksijen kültürlü 2 gün, hipoksi western blot (Şekil 1) tespit edildi HIF-1α protein bir artışa neden olur. HRE lucifeares, hipoksi kültür (embriyonik böbrek hücre hattı) 293 hücreler değiştirilen önemli bir HIF-1α aktivitesi artmış hipoksik hücreleri tespit edildi (Şekil 2). Kullanarak <img src="/files/ftp_upload/2899/2899fig1.jpg" alt="Şekil 1"/> Şekil 1: hipoksik hücrelerin HIF-1α artırın. K562 hücrelerinin 48 saat için normoxia ve hipoksi (hipoksik odasına) kültür ve HIF-1α için özel bir antikor kullanarak western blot ile analiz. Aktin için bir antikor özel yükleme kontrolü için kullanılmıştır. <img src="/files/ftp_upload/2899/2899fig2.jpg" alt="Şekil 2"/> Şekil 2: HIF-1α aktivite. HRE lusiferaz 293 hücreleri hipoksi ve 48 saat için normoxia kültüre edildi değiştirilmiş ve daha sonra bir lusiferaz tahlil kiti ve luminometre kullanarak lusiferaz sinyali tespit etmek için parçalanmış. Sonuçlar göreli lüminesans birimi (RLU) olarak ifade edilir.

<ol>
	<li>Swinson, D. E., O'Byrne, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+hypoxia+and+epidermal+growth+factor+receptor+in+non-small-cell+lung+cancer.">Interactions between hypoxia and epidermal growth factor receptor in non-small-cell lung cancer.</a> Clin Lung Cancer. 7, 250-256 (2006).</li><li>van Laarhoven, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+in+relation+to+vasculature+and+proliferation+in+liver+metastases+in+patients+with+colorectal+cancer.">Hypoxia in relation to vasculature and proliferation in liver metastases in patients with colorectal cancer.</a> Int J Radiat Oncol Biol Phys. 64, 473-482 (2006).</li><li>Hockel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intratumoral+pO2+predicts+survival+in+advanced+cancer+of+the+uterine+cervix.">Intratumoral pO2 predicts survival in advanced cancer of the uterine cervix.</a> Radiother Oncol. 26, 45-50 (1993).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF-1+and+human+disease:+one+highly+involved+factor.">HIF-1 and human disease: one highly involved factor.</a> Genes Dev. 14, 1983-1991 (2000).</li><li>Semenza, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-inducible+factor+1:+master+regulator+of+O2+homeostasis.">Hypoxia-inducible factor 1: master regulator of O2 homeostasis.</a> Curr Opin Genet Dev. 8, 588-594 (1998).</li><li>Tatum, 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=Hypoxia:+importance+in+tumor+biology,+noninvasive+measurement+by+imaging,+and+value+of+its+measurement+in+the+management+of+cancer+therapy.">Hypoxia: importance in tumor biology, noninvasive measurement by imaging, and value of its measurement in the management of cancer therapy.</a> Int J Radiat Biol. 82, 699-757 (2006).</li><li>Brahimi-Horn, M. C., Pouyssegur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIF+at+a+glance.">HIF at a glance.</a> J Cell Sci. 122, 1055-1057 (2009).</li><li>Piret, J. P., Mottet, D., Raes, M., Michiels, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CoCl2,+a+chemical+inducer+of+hypoxia-inducible+factor-1,+and+hypoxia+reduce+apoptotic+cell+death+in+hepatoma+cell+line+HepG2.">CoCl2, a chemical inducer of hypoxia-inducible factor-1, and hypoxia reduce apoptotic cell death in hepatoma cell line HepG2.</a> Ann N Y Acad Sci. 973, 443-447 (2002).</li><li>Emerling, B. M., Weinberg, F., Liu, J. L., Mak, T. W., Chandel, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTEN+regulates+p300-dependent+hypoxia-inducible+factor+1+transcriptional+activity+through+Forkhead+transcription+factor+3a+(FOXO3a).">PTEN regulates p300-dependent hypoxia-inducible factor 1 transcriptional activity through Forkhead transcription factor 3a (FOXO3a).</a> Proc Natl Acad Sci U S A. 105, 2622-2627 (2008).</li></ol>

Çıkar çatışması ilan etti.

Hipoksik koşullarda hücre proliferasyonu ve canlılığı, hücre türlerine bağlı olarak çok değişir. Bu nedenle, hücre sayısı ile başlar, deneyler için yeterli hücre / proteinlerin olacaktır emin olmak için kültürler plakaların sayısı ayarlamanız gerekir. Kobalt klorür yöntemi, ucuz ve hızlı bir avantaja sahiptir. Bu ürün HIF-1/3α uyararak hipoksi taklit eder ama bu ürün bu &quot;taklit bağımsız özel hücrelerin fonksiyon ve fenotip başka ne gibi etkileri olabilir kontrol ederek projeye adapte olduğundan emin olun, diğer genlerin düzenlenmesi hipoksik &quot;etkisi. Ayrıca hipoksi taklit etmek için kullanılan başka bir ilaç deferoksamin mesilat (DFO, 100 mcM final konsantrasyonda kullanılır). Bu ilaçların kullanımı experimentator &quot;hipoksik durum&quot; etkilemeden kültür plaka / çanak / şişesi birçok kez açmak için izin verir. Gaz karışımı bulunan oksijen düzeyi hipoksi değerleri, doku ve hücre tipleri bağlı olarak değişir deneyler ve hücre tiplerini bağlı olarak değişebilir. Gerçekten de, bazı hücreler O 2 ihtiyacı% 1&#39;den daha az O 2 hipoksik% 5 hipoksik. Kültürlerin pH stabilize etmek için% 5 CO 2 gaz karışımı eklenir, gaz geri kalanı genellikle Azot. Hipoksik odaları oksijen gerilimi bağımsız hücre davranış alternatif ilaçlar kullanarak avantajı var. Ancak, her açılışında oksijen odasına tekrar girer ve böylece hipoksi azaltır olarak her türlü deney yapılabilir. Deneylerde bu faktör göz önünde bulundurmalıdır; hipoksi / yeniden-oksijenasyon, bazı hücre tiplerinin etkileyebilir belirli bir durumdur. Alternatif bir hipoksi iş istasyonları (ön karışık gaz tankları veya gaz karışım sistemlerine bağlı) veya deney ortamı değiştirmek ve sürekli hipoksik ortamda hücreleri manipüle etmek için izin büyük hipoksi inkübatör (torpido gözü ile hipoksi işlem odası) kullanmaktır. Çeşitli hipoksik odaları son on yıl içinde satışa sunulmuştur ve sizin laboratuar, proje, uzay, boyut, ve bütçe göre seçilmelidir. Her zaman doğru kültür koşulları garanti altına almak için odasının iyi kullanmak ve durumu sağlamak. Genel olarak bir oda kullanırken, hipoksi düzeyi çeşitli gaz karışımları (yani% 1,% 5,% 10 oksijen) seçerek modüle edilebilir. HIF-1α tespiti ile ilgili olarak, bilmek önemlidir ki, bazı kanser hücre hattı normoxia olarak ifade HIF-1α. Bu nedenle, bu hücre hatları HIF bazal düzeyini belirlemek için bir normoxia kontrol kullanmak için kritik öneme sahiptir. HIF-1α hücre stimülasyon veya stres sonrası non-malign hücrelerin normoxia de mevcut olabilir. Bu aynı zamanda, bu hücreleri aç meydana hücre kültürleri doğru beslenmek ve korunur emin olun.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Anti-HIF-1α </td><td> Invitrogen </td><td> 458400 </td></tr><tr><td> Kobalt (II) Klorür </td><td> Sigma </td><td> C8661-25G </td></tr><tr><td> İnkübatör Odası </td><td> Billups-Rothenberg </td><td> MIC-101 </td></tr><tr><td> HRE-Lusiferaz plazmid </td><td> Adgene </td><td> 26731 Plazmid </td></tr></tbody></table>

induction and testing of hypoxia in cell culture

Hipoksi, organ, doku veya hücrelerin oksijen azalması veya yokluğu olarak tanımlanır. Bu azalma, oksijen gerginlik azalmış oksijen kaynağı (yetersiz kan damarı ağı, arızalı kan damarı ve anemiye neden olur) ya da tedarik göre (ani bir yüksek hücre proliferasyon oranı neden) oksijen tüketiminin artmasına bağlı olabilir . Hipoksi, 1-3, romatoid artrit, ateroskleroz, vb katı kanserleri gibi fizyolojik veya patolojik olabilir ... Her doku ve hücrelerin bu yeni duruma uyum sağlamak için farklı bir yeteneği var . Hipoksi sırasında, hipoksi indüklenebilir faktör alfa (HIF) stabilize ve oksijen 4 anjiyogenez veya nakliye dahil olanlar gibi çeşitli genler düzenler. Bu nedenle, bu proteinin istikrar HIF rutin hipoksi 5-7 için ekran için kullanılır tespit, hipoksi bir özelliğidir. Bu makalede, biz, bu hücrelerin hipoksik durumunu değerlendirmek için memeli hücre kültürleri ve basit testler hipoksi ikna etmek için iki basit yöntem öneriyoruz.

Watch this Scientific Journal Video about Induction and Testing of Hypoxia in Cell Culture at JoVE.com

Induction and Testing of Hypoxia in Cell Culture

Burada kültürlerin hipoksik durumunu değerlendirmek için, hücre kültürleri ve basit testler hipoksi ikna etmek için basit yöntemler öneriyoruz.

Hücre Kültürü ve Hipoksi Test İndüksiyon

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

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Research

JoVE Journal

Biology

84.4K Görüntüleme.  Baylor College of Medicine. Burada kültürlerin hipoksik durumunu değerlendirmek için, hücre kültürleri ve basit testler hipoksi ikna etmek için basit yöntemler öneriyoruz.

Video: Induction and Testing of Hypoxia in Cell Culture

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Fare Nöral Kök Hücre Öncüleri Kültür

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Biyoloji

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.

Sıçanlarda Kabul edilmiş Gecikmeli-Tipi aşırı duyarlılık indüksiyonu ve İzleme

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Sıçan Karaciğer Hücreleri İzolasyon ve İlköğretim Kültür

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Eksplantasyon Kültüründe Diş mikropların Geliştirme takiben için Dilim Kültür Yöntemi

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Kas Uydu Hücreleri İzolasyon, Kültür ve Transplantasyon

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

c-FOS Protein Immünohistolojik Algılama: Belirli Fizyolojik Yanıtları ilgilendim Merkez Pathways bir Marker Olarak Faydalı Aracı<em&gt; İn Vivo</em&gt; ve<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA bağlama proteinlerinin tespiti ile<em&gt; İn Vitro</emAdiposit Kültür&gt; RNA pull-down

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Deniz Hıyar Bağırsak Hücrelerinin Birincil Kültüründe Apoptosis İndüksiyon uyupve tayini

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

Methods Article

Hücre Kültürü ve Hipoksi Test İndüksiyon