Bu çalışma Emniyeti&#39;ne Emniyeti&#39;ne Amerikan Kalp Derneği ödül 13SDG14500046, Sürdürülebilir Enerji Eğitim ve Araştırma Merkezi Ödülü (Tennessee Univ.) tarafından desteklenen ve JM için Hekim Tıp Eğitimi ve Araştırma Vakfı (Tennessee Univ.) tarafından ödüllü edildi Onun sallayarak inkübatör, masaüstü santrifüj, ve Western Blot analizi ekipman kullanımı için Eric T. Boder (Tennessee Univ.); yazarlar spheroplastlar için fisyon maya dönüştürmek için protokol için Caroline Leplante (. Yale Üniversitesi) teşekkür ve için Merkezi ultra santrifüj kullanımı için Çevresel Biyoteknoloji (Univ. Tennessee); maya antikorlar Günther Daum (Graz Üniversitesi Teknoloji, Avusturya.); teknik yardım için Kadın Hastalıkları ve Doğum (Univ. Tennessee Tıp Merkezi) Bölümü personel .

1.. (Fisyon) Maya Hücreleri gelen Lipid Damlalarda Yalıtımlı Maya Saccharomyces cerevisiae tomurcuklanan popüler model organizma damlacıkları izolasyonu aşağıdaki protokole 6 hemen hemen aynıdır. Hazırlıklarında farklılıklar belirtilmiştir. 1.. Büyüyen maya hücreleri <ol><li> Ortamını hazırlayın. Cam şişe ya da kültür şişelerinde dH 2 O litresi başına YE5S toz 36 g &#39;ını. Ortam yaklaşık 2 L gerekli olacaktır. 20 dakika boyunca 121 ° C&#39;de otoklav ortamı. Medya oda sıcaklığına kadar soğumasını bekleyiniz. S. cerevisiae YDP ile YE5S değiştirin. </li><li> Steril teknikler kullanılarak, 250 ml kültür şişesi içine soğutulmuş YE5S ortamın 10-20 ml yerleştirin. Steril bir tahta bir çubuk ya da eşdeğeri kullanılarak Bir agar plakasından maya hücrelerinin küçük bir miktar ile inoküle ortamı. Hücreler, 30 ° C&#39;de, istenen optik yoğunluğa büyümesine izin A optik yoğunluğunun ölçülmesiBir spektrofotometre kullanılarak 595 nm dalga boyu. </li><li> 2.8 L şişelerinde kalan ortamı yerleştirin. Çalkalama inkübatöründe hücre büyümesi sırasında düzgün karışmasını sağlamak için ortam başına 2.8 L şişenin en fazla 1 L kullanın. Islak hücre Nihai verim proteomik ve lipidomic analiz için yeterli lipid damlacıkları elde edebilmek için yaklaşık 10 g olmalıdır. </li><li> Her bir adım 1.1 hazırlandı ortamı 1 L olan 2.8 L şişelere aşama 1.2 &#39;den hücreleri tanıtılması. </li><li> 30 ° C&#39;de arzu edilen yoğunluğa hücrelerin büyümesine </li><li> Hücrelerin lipid damlacıkları olduğundan emin olun. Hücrelerin Reserve 1 mi. Numunenin optik yoğunluğu (OD) ölçün. Numuneye hücrelerin OD başına etanol içinde BODIPY 493/503 içinde bir 100 mM stok 0.1 ml ilave edilir. Bir cam slayt ve bir cam kapak kayma arasındaki örnek 3 ul koyun. GFP filtre (Şekil 1A) ile bir floresan mikroskop altında gözünüzde canlandırın. </li><li> 4 ve # &#39;de aşama 1.5 gelen hücreleri Pelet176, C 10 dakika boyunca 3,800 x g. Santrifüj tüplerinin kapasitesine bağlı olarak gruplar halinde çalışır. </li><li> YE5S ortamı dökün ve sorbitol EMM + 600 mM tampon maddesi ile hücreleri yıkayın. Sorbitol ozmotik destek sağlar. </li><li> , Steril bir 50 ml santrifüj tüpüne hücreleri aktarın ve 10 dakika boyunca 2000 x g&#39;de 4 ° C&#39;de pellet hücreleri. Süpernatant kaldırmak. Islak hücreler tartılır. Hücre gramı başına (EMM + 600 mM sorbitol), tampon 2 ml hücreleri yeniden süspanse edin. </li></ol> 2. Spheroplastlar ve daha sonra lizis içine bölünmüş maya hücreleri dönüştürme <ol><li> ) Maya litik enzim 5 mg 1 Her ekleyin ve 2) maya Trichoderma harzianum başına gram adımda 1.9 tartıldı ıslak hücre enzimleri yokedici. S. cerevisiae askiyle sonuçlanır ki aynı miktarda bu enzimleri artırmaktadır. </li><li> 60 dakika boyunca 100 rpm&#39;de hafif sallama altında 30 ° C&#39;de inkübe hücreleri. </li><li> Sferoplastlar lipit dr emin olmak için kontrol edinoplets. Adım 1.6 (Şekil 1 B) tekrarlanarak. </li></ol> 3. Density gradient santrifüjleme <ol><li> 5 dakika boyunca 2000 x g&#39;de 4 ° C&#39;de santrifüj ile spheroplastlar Pelet. </li><li> Dikkatle plastik bir transfer pipet kullanarak süpernatant kaldırmak. </li><li> Yavaşça buz gibi soğuk 10 mM Tris-HCI, 600 mM sorbitol, ve 200 uM EDTA ile pelet spheroplastlar tekrar süspansiyon. Sferoplastlar çok kırılgan olduğunu aklınızda tutun. </li><li> Adım 3.1 olarak aynı ayarları kullanarak yeniden spheroplastlar Pelet. Süpernatantı dikkatlice çıkarın ve 2 ml / g-hücre konsantrasyonunda 12% Ficoll plastik transfer pipeti, 10 mM Tris-HCl ve 200 mM EDTA kullanılarak spheroplastlar tekrar süspansiyon. </li><li> Bir cam homojenizatör üzere yeniden süspansiyon haline getirilmiş ve yavaşça spheroplastlar transfer cam homojenleştirici gevşek havan tokmağı ile, buz üzerinde 20-30 vuruş homojenleştirin. </li><li> Santrifüj tüplerine parçalanır spheroplastlar aktarın ve aynı vo kaplanacak% 12 Ficoll, 10 mM Tris-HCl ve 200 uM EDTA tamponu lume. Böylece her tüp yaklaşık üçte ikisi dolu olduğunu kullanmak için santrifüj tüplerinin sayısını seçin. </li><li> Bir SVV28 rotor veya eşdeğeri, 1.5 saat boyunca 100,000 x g hızında, 4 ° C &#39;de santrifüj. Bir stop (rotor yavaşlama = 0) için kıyıya rotor izin verin. </li><li> Yavaşça bir pipet ya da bir bükülmüş Pasteur pipeti kullanarak yeni bir santrifüj tüpüne (ler) için damlacıkları içeren üst yüzen beyaz tabaka (Şekil 2A), transfer. Bu santrifüj tüpünün aşağı yukarı üçte biri doldurur, böylece numune için yeterli% 12 Ficoll hacmi, 10 mM Tris-HCl ve 200 uM EDTA tamponu ekleyin. </li><li> Yeni bir santrifüj tüpüne (s) &#39;de örnek (ler) üstüne denk% 8 Ficoll hacmi, 10 mM Tris-HCl ve 200 uM EDTA tamponu ekleyin. Bu eklenmesinden sonra, tüpler tam üçte ikisi olmalıdır tampon. </li><li> Bir SVV28 rotor veya eşdeğeri 1 saat boyunca 100,000 x g hızında, 4 ° C &#39;de santrifüj. Durdurmak için kıyıya rotor izin (rotor yavaşlama = 0). </li><li> Yavaşça bir pipet ya da bir bükülmüş Pasteur pipeti kullanarak yeni bir santrifüj tüpüne (ler), damlacıkları içerecek En iyi yüzen beyaz tabaka (Şekil 2B), aktarın. Bu santrifüj tüpüne yaklaşık üçte biri doldurur, böylece örnek, sorbitol% 8 Ficoll, 10 mM Tris-HCl ve 200 mM EDTA tampon maddesi 600 mM ekleyin. </li><li> Yeni bir santrifüj tüpüne (s) &#39;de örnek (ler) en üstüne, sorbitol 10 mM Tris-HCl, 250 mM eşdeğer hacim ekleyin ve 200 uM EDTA tampon maddesi. Bu eklenmesinden sonra, tüpler tam üçte ikisi olmalıdır tampon. </li><li> Bir SVV28 rotor veya eşdeğeri 1 saat boyunca 100,000 x g hızında, 4 ° C &#39;de santrifüj. Bir stop (rotor yavaşlama = 0) için kıyıya rotor izin verin. </li><li> Diyaliz boru için, sadece lipid damlacıkları ve bağlanmış proteinler içerir ve Ficoll ortadan kaldırmak için 10 mM Tris-HCl ve 200 mM EDTA tampon olarak O / N dialyze gerektiği iyi beyaz bir tabaka (Şekil 2C), aktarın. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. İnsan Plasental Villöz Hücreler Lipid Damlalarda Yalıtımlı Plasenta tekiz gebelik öncesi dönemde spontan doğumun başlamasından elektif sezaryen doğum yapan ile sağlıklı kadınlarda toplanmıştır. Denekler kendi plasenta koleksiyonu için yazılmış, bilgilendirilmiş onam verdi. Plasentaların toplanması ve daha sonraki kullanım, Knoxville Kurumsal Değerlendirme Kurulu Tennessee ve Tıp Tennessee Enstitüsü Üniversitesi Üniversitesi (sırasıyla # # 8757B ve 3338) onayı ile yapıldı. 1.. Insan plasental villöz hücreleri tecrit Protokolün Part 1 Petroff et al. 26 daha önceki bir yayınlanan bir protokolün modifikasyonudur Aşağıdaki çözeltiler hazırlayın: <ul><li> NaCI (1 L), bir 1 L&#39;lik bir çözelti elde etmek dH 2 O 9 g NaCl (M + 58.4 g / mol) içinde çözülür. Filtre sterilize (0.2; Mikron zar). </li><li> 10x Hank dengeli tuz çözeltisi (10x HBSS) (1 L): 4 g KCl (M + 74.5 g / mol), 0.6 g KH 2 PO 4 (M + 136,086 g / mol), 80 g NaCl (M 58,44 g / mol); 10 g D-glukoz (M 180,16 g / mol), 2 HPO 4 (M + 141.96 g / mol) Na. Bir 1 L çözelti elde etmek üzere dH 2 O bileşenlerin her çözülür. Filtre sterilize (0.2 mikron zar). </li><li> Enzim sindirim tamponu (0.6 mi) 3.3 ml% 7.5 Na Biscarbonate, 17.5 ml 1 M HEPES, ve 266.1 ml dH 2 O ile 70 ml HBSS 10x birleştirmek, sterilize (0.2 mikron zar). 4 ° C&#39;de saklayın </li><li> DNase I: Kullanımdan hemen önce, DNase bir şişeye, 5 ml steril% 0.9 NaCl ilave edin. -20 ° C&#39;de kullanıma veya mağaza kadar buz üzerinde tutmak </li></ul><ol><li> Diseksiyon için hazırlık plasenta <ol><li> Bir insan plasenta ve mümkün olduğunca teslimat zamanına yakın süreci edinin. Bu tür plasenta taşımak için bir soğutucu olarak bir sızdırmaz kılınmış tekne kullanın. Zaman el her zaman dikkatli olunling insan biyolojik materyaller. </li><li> Biyolojik güvenlik kabini içinde, steril bir otoklavlanabilir bir kaba aktarmak plasenta ve dikkatli bir şekilde steril% 0.9 NaCl (tuzlu su) çözeltisi kullanılarak plasenta ve membranlar kan yıkayın. Sıvı biyolojik atık olarak 1 L beher kanlı tuz atın. </li><li> Pürüzsüz yüzey yukarı bakacak göbek kordonunu rulman ile, steril bir alan üzerinde plasentayı yerleştirin. Keskin, ince nokta makas ve forseps ile fetal membranlar ve göbek kordonunu çıkarın. </li><li> Maternal yüzey (pürüzlü yüzey) yukarıya bakacak şekilde plasentayı ters çevirin. Örten bazal plaka doku, yüzeyden yaklaşık 3 mm çıkarın. </li></ol></li><li> Plasenta Diseksiyon <ol><li> Koryon plaka kaçınarak her seferinde bir kotiledon ayır. Tuzlu su ile 250 ml&#39;lik bir behere villöz doku toplayın. </li><li> Sıvı atık için 1 L kabı kullanarak, forseps ile dönen ile% 0.9 NaCI ile ayrı bir çanak içinde doku birkaç kez yıkayın. </li><li> Bir 150 mm Petri kabı, bir seferde bir kotiledon aktarın. Forseps ile tutun ve Petri kabı gemilerin jilet ile doku kazıyın. % 0.9 NaCl içeren ayrı bir beher içine kazınmış doku yerleştirin. Tüm doku parçaları için tekrarlayın. </li><li> Hücre ayrışma eleğe doku küçük bir bölümünü transfer ve ortaya çıkan ürün berrak hale gelinceye kadar yaygın olarak% 0.9 NaCl ile yıkayınız. Her doku için tekrarlayın. </li><li> Hücre ayrışma elek kazınmış doku durulanmasından sonra, fazla sıvı ve ağırlık olarak boşaltın. Bu noktada doku DMEM içinde 4 ° C&#39;de steril bir Erlenmeyer şişesi içinde O / N depolanabilir. </li></ol></li><li> Plasenta dokusundan enzimatik sindirim <ol><li> Önceden ısıtılmış enzim seyreltme tamponu 100 mi, 1 ml DNase I ve 20 ml% 2.5 tripsin 10x: Doku sindirimi karışım hazırlayın. </li><li> 500 ml&#39;lik bir Erlenmeyer şişeye steril toplanan toplam doku (genellikle yaklaşık 250-300 g), 60 g aktarılması, doku bölün. </li><li&gt; doku içeren Erlenmeyer şişeye doku sindirim karışımı ekleyin ve 150 rpm&#39;de 45 dakika için bir sallayıcı içinde 37 ° C&#39;de inkübe edin. </li></ol></li><li> Ayrışmış hücreleri toplamak <ol><li> Doku yerleşir kadar ilk 45 dakika ayrıldıktan sonra, bir eğimde sindirim şişeyi ayarlayın. </li><li> Ayrışmamış doku toplamak için özen, süpernatant toplayın. Toplanan yüzer için HBSS eşit miktarda eklenir ve steril bir 15 ml santrifüj tüplerine aktarılır. Çözünmemiş doku malzemesinden ek kısımlar için, çözülmemiş doku ile adım 3.2 başlangıç ​​prosedürü tekrarlayın. </li><li> 15 dakika boyunca 1000 x g&#39;de 4 ° C&#39;de Homojenat santrifüj. </li><li> Her grup için, santrifüjün ardından, pelet bozmadan, süpernatan aspire. Plasental villöz hücreler, kırmızı kan hücreleri üzerinde uzanan, pelet beyaz bölümünde ağırlıklı olarak. </li><li> Bir st villus hücreleri (pelet beyaz kısmı) aktarın50 mL konik santrifüj tüpü erile ve buz üzerinde tutun. </li><li> Üç sindirim aşamalarında hücreleri toplanmasından sonra, steril bir 50 ml lik konik bir santrifüj tüpünün üst sokulan bir 100 um naylon hücre süzgeç kullanılarak süspansiyon filtre. Hücre süspansiyonunun filtrasyon yavaşlattığında, boru içinde bir vakum çekmek için filtre üzerinde yukarı kaldırın. </li><li> 10 dakika boyunca 1000 x g&#39;de 4 ° C&#39;de santrifüje. Dikkatle pelet bozmadan süpernatant kaldırmak. </li></ol></li></ol> 2. Plasental villöz hücreleri homojenize <ol><li> Izolasyon işlemi başlamadan önce, 20 mM Tris-HCI, pH 7.4 ve 1 mM EDTA ihtiva eden Hipotonik Lysis Ortamı (HLM) 50 ml hazırlar. Kullanımdan hemen önce HLM proteaz inhibitörleri eklenir ve buz üzerinde orta tutun. </li><li> Biyolojik güvenlik kabini olarak, hücrelere buz soğukluğunda HLM 4 katı hücre topağı ses ekleyin. Yavaşça ve iyice hücreleri pipetleme hücreleri tekrar süspansiyon yukarı bir10 ml pipet kullanarak d-aşağı. </li><li> 10 dakika boyunca buz üzerinde asılı hücreleri inkübe edin. </li><li> Buz üzerinde olmalıdır Dounce homojenleştirici, hücreleri aktarın. Yavaş yavaş homojenleştirici gevşek havan tokmağı ile 20-25 hafif vuruş uygulayarak hücreleri homojenize edilmiştir. </li><li> Santrifüj 10 dakika boyunca 3000 x g, 4 ° C &#39;de, hücre lisatı kesintisiz hücreler, hücre artıkları ve çekirdeklerin giderilmesi için. </li><li> Süpernatant toplayın ve bir SVV28 tüp (ya da 25.000 x g&#39;de santrifüj edilebilir alternatif) transfer. Mitokondri çıkarmak için 20 dakika için 25,000 xg&#39;de 4 ° C&#39;de santrifüj. </li></ol> 3. Ultra-santrifüj ile izole edilmesi lipid damlacıkları <ol><li> 50 mM sodyum karbonat, 100 (M + 106 g / mol) tamponu (pH 11.5) ve ml ve 50 mi% 60 sukroz (ağırlık / ağırlık) çözeltisi hazırlayın. Kullanımdan hemen önce, sodyum karbonat tamponuna proteaz inhibitörleri eklenir ve buz üzerinde tutun. </li><li> Ayarlayın, bir 50 ml santrifüj tüpüne aşama 2.6 &#39;dan süpernatan toplamakBir SVV28 rotoru ya da eşdeğer bir ultra santrifüj tüpünün dibine sükroz ve transfer% 20. ~ Buz gibi soğuk 100 mM sodyum karbonat tamponu (pH 11.5) 10 ml ve tüpün doldurulması için buz soğukluğunda HLM 0.5-1 ml Kaplama yoğunluğu ayarlanabilir yüzer. </li><li> SVV28 sallanan kovalı rotor kullanılarak 45 dakika boyunca 130,000 x g&#39;de, 4 ° C&#39;de santrifüj. Bir stop (rotor yavaşlama = 0) için kıyıya rotor izin verin. </li><li> % 10 sakaroza ayarlamak ve bir SW41Ti rotor ya da eşdeğer bir 13.2 ml ultra santrifüj tüpünün tabanına içine aktarın, yüzen tabakayı toplayın. Kaplama yoğunluğu yaklaşık 5 buz gibi soğuk 100 mM sodyum karbonat tamponu (pH 11.5) içinde çözdürülür ve buz soğukluğundaki HLM 0.5 ml süpernatant ayarlandı. </li><li> SW41Ti sallanan kovalı rotor kullanılarak 60 dakika boyunca 274,000 x g&#39;de, 4 ° C&#39;de santrifüj. (Rotor yavaşlama = 0) lipit damlacık tabakasının bozulmasını en aza indirmek için bir durdurmak için kıyıya rotor izin verin. </li><li> Dikkatle içeren üst yüzen tabaka (Şekil 2B) toplamak1 ml pipet ve tekrar adım 3.4 ile lipid damlacıkları. </li><li> SW41Ti sallanan kovalı rotor kullanılarak 30 dakika boyunca 274,000 x g&#39;de, 4 ° C&#39;de santrifüj. Bir stop (rotor yavaşlama = 0) için kıyıya rotor izin verin. </li><li> 100&#39;yi ul Hlm içeren üç 1.5 ml tüpler içine bükülmüş Pasteur pipeti ile lipid damlacıkları içeren üst beyaz renkli kayan tabaka toplamak. </li></ol> Niteleme, lipid damlacıkları fraksiyonu (Protokoller 1 ve 2) Lipid damlacık fraksiyonunun geri kazanımı ve saflık ışık veya elektron mikroskobu ile birlikte Western Blot ile doğrulanabilir. Buna ek olarak, farklı santrifüj adımları sonra tam bölünen miktarları, toplanabilir ve arıtma verimi belirlemek için korunmuştur. Western Blot, bu t diğer zar fraksiyonları lipid damlacıkları karşılaştırarak daha bütün hücre lizatının bir eşdeğeridir lipit fraksiyonunun bir damlacık hacmi karşılaştırmak için daha uygun olduğunutoplam protein içeriği 24 diye temeli.

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L., Dolios, G., Shapiro, L., Wang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+proteins+associated+with+lipid+droplets+of+basal+and+lipolytically+stimulated+3T3-L1+adipocytes.">Proteomic analysis of proteins associated with lipid droplets of basal and lipolytically stimulated 3T3-L1 adipocytes.</a> J. Biol. Chem. 279, 46835-46842 (2004).</li><li>Liu, P. S., Ying, Y. S., Zhao, Y. M., Mundy, D. I., Zhu, M. F., Anderson, R. G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chinese+hamster+ovary+K2+cell+lipid+droplets+appear+to+be+metabolic+organelles+involved+in+membrane+traffic.">Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic.</a> J. Biol. Chem. 279, 3787-3792 (2004).</li><li>Fujimoto, 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=Identification+of+major+proteins+in+the+lipid+droplet-enriched+fraction+isolated+from+the+human+hepatocyte+cell+line+HuH7.">Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.</a> Biochim. Biophys. Acta. , 47-59 (2004).</li><li>Sato, S., 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=Proteomic+profiling+of+lipid+droplet+proteins+in+hepatoma+cell+lines+expressing+hepatitis+C+virus+core+protein.">Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.</a> J. Biochem. 139, 921-930 (2006).</li><li>Umlauf, E., Csaszar, E., Moertelmaier, M., Schuetz, G. J., Parton, R. G., Prohaska, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+stomatin+with+lipid+bodies.">Association of stomatin with lipid bodies.</a> J. Biol. Chem. 279, 23699-23709 (2004).</li><li>Zhang, H. N., 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=Proteome+of+skeletal+muscle+lipid+droplet+reveals+association+with+mitochondria+and+apolipoprotein+A-I.">Proteome of skeletal muscle lipid droplet reveals association with mitochondria and apolipoprotein A-I.</a> J. Proteome Res. 10, 4757-4768 (2011).</li><li>Wu, C. C., Howell, K. E., Neville, M. C., Yates, J. R., McManaman, J. 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P., 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=Lipid+droplet+de+novo+formation+and+fission+are+linked+to+the+cell+cycle+in+fission+yeast.">Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast.</a> Traffic. 13, 705-714 (2012).</li><li>Petroff, M. G., Phillips, T. A., Ka, H., Pace, J. L., Hunt, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+term+human+trophoblast+cells.">Isolation and culture of term human trophoblast cells.</a> Methods Mol. Med. 121, 203-217 (2006).</li><li>Cho, S. 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=Identification+of+mouse+Prp19p+as+a+lipid+droplet-associated+protein+and+its+possible+involvement+in+the+biogenesis+of+lipid+droplets.">Identification of mouse Prp19p as a lipid droplet-associated protein and its possible involvement in the biogenesis of lipid droplets.</a> J. Biol. Chem. 282, 2456-2465 (2007).</li><li>Ding, Y. B., Wu, Y. B., Zeng, R., Liao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+profiling+of+lipid+droplet-associated+proteins+in+primary+adipocytes+of+normal+and+obese+mouse.">Proteomic profiling of lipid droplet-associated proteins in primary adipocytes of normal and obese mouse.</a> Biochim. Biophys. Acta. 44, 394-406 (2012).</li><li>Jagerstrom, S., 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=Lipid+droplets+interact+with+mitochondria+using+SNAP23.">Lipid droplets interact with mitochondria using SNAP23.</a> Cell Biol. Int. 33, 934-940 (2009).</li><li>Xu, N. 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=The+FATP1-DGAT2+complex+facilitates+lipid+droplet+expansion+at+the+ER-lipid+droplet+interface.">The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface.</a> J. Cell Biol. 198, 895-911 (2012).</li><li>Ozeki, S., Cheng, J. L., Tauchi-Sato, K., Hatano, N., Taniguchi, H., Fujimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rab18+localizes+to+lipid+droplets+and+induces+their+close+apposition+to+the+endoplasmic+reticulum-derived+membrane.">Rab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane.</a> J. Cell Sci. 118, 2601-2611 (2005).</li><li>Yang, H. Y., Galea, A., Sytnyk, V., Crossley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+size+of+lipid+droplets:+lipid+and+protein+factors.">Controlling the size of lipid droplets: lipid and protein factors.</a> Curr. Opin. Cell Biol. 24, 509-516 (2012).</li><li>Jacquier, N., Choudhary, V., Mari, M., Toulmay, A., Reggiori, F., Schneiter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+droplets+are+functionally+connected+to+the+endoplasmic+reticulum+in+Saccharomyces+cerevisiae.">Lipid droplets are functionally connected to the endoplasmic reticulum in Saccharomyces cerevisiae.</a> J. Cell Sci. 124, 2424-2437 (2011).</li><li>Pu, J., Ha, C. W., Zhang, S., Jung, J. P., Huh, W. K., Liu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactomic+study+on+interaction+between+lipid+droplets+and+mitochondria.">Interactomic study on interaction between lipid droplets and mitochondria.</a> Protein Cell. 2, 487-496 (2011).</li><li>Shaw, C. S., Jones, D. A., Wagemakers, A. J. 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Chem. 287, 2273-2279 (2012).</li></ol>

Yazarlar herhangi bir mali çıkarlarını beyan.

<html> Bu protokol kapsamında kritik adım Kültür haline gelmiş hücrelerin büyümesi sırasında ortam ve hücre yoğunlukları ile tutarlı olması için emin olun. Cellular lipid damlacıkları, ilişkili proteinlerin hücreler kültürlendi 17 olduğu çevre üzerine son derece bağımlı olmasıyla benzersizdir. Bu nedenle, hücrelerin yetiştirildiği ortam ve hücre yoğunluğu yakından lizis önce izlenmelidir. <p class="jove_cont...

Hücresel lipid damlacıkları hücrelerinde birden fazla işleve hizmet dinamik organelleri. Bunlar enerjiye dönüştürülür ya da fosfolipid sentezi için kullanılabilir nötr lipidler, depolama merkezleri vardır. Damlacıklar, ateroskleroz, obezite ve ilişkili metabolik hastalıklar da dahil olmak üzere fizyolojik ve patolojik durumlarda merkezi bir rol oynar ve aynı zamanda bulaşıcı hastalıklar 1,2. Buna ek olarak, biyodizel yakıtları için ilgi çekici kaynaklarıdır. Lipid damlacıkları hücre rolleri hakkında bilgisi geniş organizmalar 3 saflaştırılan damlacıklarının proteomik ve lipidomic analizinden elde edilmiştir. Bu organizmalar, bakteri 4,5, maya 6-11 dahil 12,13 bitkiler, 14 nematodları, ve 15,16 sinekler var. İnsan metabolik hastalıklar lipid damlaları rolü olan ilgi dikkate alındığında, damlacıkları ile kültürden geçirilmiş hayvan hücreleri ve a izole edilmişNimal dokular. Kültürlü hücre çizgileri, 3T3-L1 adipositler 17, Çin hamsteri yumurtalık (CHO) hücreleri 18 K2, insan hepatocyes 19,20 ve epitel hücre hatları 21 dahil ettik. Damlacıklar izole edilmiş olan hayvan dokuları fare iskelet kas 22, karaciğer 23 ve 23 meme bezleri dahil ettik. Yukarıda zikredildiği gibi, en çok damlacık izolasyon çalışmaların amacı bağlı faktör ile nötr ve fosfolipidlerden lipidomic analizine proteomik analiz etmektir. Nötr lipidler yana - lipid damlacıkları en çok bileşenli - En çok diğer hücre malzemelere nazaran daha az yoğun olan, damlacıkların izolasyon geleneksel yoğunluk gradyan santrifüjlemesi kullanılarak gerçekleştirilmiştir. Bu tekniği Burada sunulan iki preparatlarıyla merkezinde olduğunu. Önceki teknikler 6,24 kültürlenmiş bölünmüş maya hücrelerinden damlacıklarının izolasyonu bir görsel sunum halinde bir araya getirilmiş ve modifiyeve noncultured insan hücreleri plasenta dokusundan elde edilmiştir. Amaç damlacık izolasyonu için başlangıç ​​noktası olarak, iki çok farklı hücre tiplerini seçerek bu tekniğin geniş uygulanabilirlik göstermektir. Bu teknik çoğu organizmalar damlacıkların izole etmek isteyenler için yararlı olacaktır. Protokol 1 ökaryotik hücre bölünmesi esnasında 25, damlacık oluşumunu gözlemlemek için bir model olarak kullanılmıştır fizyon maya, Schizosaccharomyces pombe, ikinci lipid damlacıkları izolasyonunu tarif eder. Tomurcuklanan maya Saccharomyces cerevisiae lipid damlacık biyoloji eğitimi için bir model organizma olarak yaygın şekilde kullanılmaktadır. Protokol 1 organizmalar ve hazırlıkları farklılıklar hem de uygulanabilir vurgulanır. Protokol 2 İnsan plasentası terimi, elde edilen sırayla olan plasental villöz hücrelerden lipid damlacıkları izolasyonunu tarif eder.Terim Plasentaların koleksiyonu güvenle ve etik lipid damlacıkları önemli numaralarını içeren hazır insan dokusunda 26, 200-250 g almak için eşsiz bir fırsat sunuyor. Bu damlacıklar kültürlenmiş hücrelerden kaynaklanan daha yüksek ökaryotlarda en lipid damlacık izolasyon çalışma aksine bulunmaktadır. Bu çalışmalarda, yağ asitleri genellikle nötral lipid ve böylece damlacıkların büyümesinin sentezini teşvik etmek için kültüre eklenir. Bu lipid damlacıkları plasental dokudaki doğal koşullar altında oluşturulmaktadır burada çalışma aksine bulunmaktadır. Lipid damlacık fraksiyonlarının saflıkları organel markör antikorları kullanılarak Western Blot analizi ile belirlenir. Bu iki protokol sonraki proteomik ve lipidomic analiz için uygun olan lipid damlacık kesirler verecektir.

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			<td height="21" style="height:21px;width:353px;">PROTOCOL #1:&#160;</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:353px;">1.Growing yeast cells and converting to spheroplasts</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Edinburgh Minimal Media (EMM)</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">2005</td>
			<td></td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;">Yeast extract with 5 supplements (YE5S)</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">2011</td>
			<td style="width:167px;">YE5S media with 225 mg/ml of each supplement: adeninie, histidine, leucine, lysine, uracil. The equivalent for budding yeast would be YPD.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">YPD powder</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">1875</td>
			<td>For S. cerevisiae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sorbitol</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP439</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Yeast Lytic Enzyme</td>
			<td>MP Biomedicals</td>
			<td style="width:119px;">215352610</td>
			<td></td>
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			<td height="20" style="height:20px;">Lysing Enzymes from Trichoderma harzianum</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">L1412</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zymolayse-20T</td>
			<td>Sunrise Science Products</td>
			<td style="width:119px;">N0766391</td>
			<td>For S. cerevisiae&#160;</td>
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			<td height="20" style="height:20px;">BODIPY 493/503</td>
			<td>Invitrogen</td>
			<td style="width:119px;">D-3922</td>
			<td></td>
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			<td height="20" style="height:20px;">Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">12-544-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope Cover Glass</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">12-542-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic transfer pipette</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">137115AM</td>
			<td></td>
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			<td height="21" style="height:21px;">1 liter glass bottle</td>
			<td></td>
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			<td height="21" style="height:21px;">250 ml flask</td>
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			<td height="21" style="height:21px;">2.8 liter flasks</td>
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			<td height="20" style="height:20px;">2. Yeast lipid droplet isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll 400</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP525</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-14k Spectra/Por Dialysis Membrane</td>
			<td>SpectrumLabs</td>
			<td style="width:119px;">132680</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free Protease Inhibitor Cocktail Tablets</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11873580001</td>
			<td>irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce Homogenizer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Tubes 25x89mm (for SW28)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">355642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12-14k Spectra/Por Dialysis Membrane</td>
			<td>SpectrumLabs</td>
			<td style="width:119px;">132680</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Temperature-controlled shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;">C25KC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Sorvall Legend XTR centrifuge</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75004521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swinging Bucket Centrifuge Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiberlite* F15-6x100y Fixed-Angle Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003698</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROTOCOL #2&#160;</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1. Placental villous cells isolation</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable underpads</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">23666062</td>
			<td style="width:167px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Autoclavable pan (container), 3L</td>
			<td style="width:163px;">Fisher Scientific</td>
			<td style="width:119px;">1336110</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fine scissors, sharp-sharp, straight</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1406011</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">London Forceps</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1108002</td>
			<td style="width:167px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Dumont #7b Forceps</td>
			<td>Fine science tools</td>
			<td style="width:119px;">1127020</td>
			<td style="width:167px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Razor blades</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S65921</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Screen cup for CD-1</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S1145</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40 mesh screen&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S0770</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand cell stainers 100&#956;m</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">22363549</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">150 mm Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">NC9054771</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S642</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">P333</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">P386</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">S374</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-glucose</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">D16</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.5% trypsin 10x</td>
			<td>Invitrogen</td>
			<td style="width:119px;">15090046</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNase I grade II, from bovine pancreas</td>
			<td>Roche Applied Science</td>
			<td style="width:119px;">10104159001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium bicarbonate solution</td>
			<td>Sigma-aldrich</td>
			<td style="width:119px;">S8761</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">500 ml Erlenmeyer flasks</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">250 ml beakers</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">15 ml centrifuge tubes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">10 ml serological pipettes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50 ml centrifuge tubes</td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">DMEM</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">11965084</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2. Lipid droplets isolation from villous placental cells</td>
			<td></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-Sucrose</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Carbonate&#160;</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">BP357</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA-free protease inhibitor cocktail tablets</td>
			<td>Roche Diagnostics</td>
			<td style="width:119px;">11873580001</td>
			<td>irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dounce homogenizer&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:119px;">D9938</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge tubes 25x89mm (for SW28)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">355642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-Clear centrifuge tubes 14x89mm (for SW41)</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">344059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable borosilicate glass pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;">1367820C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Equipment</td>
			<td style="width:163px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Biological safety hood&#160;</td>
			<td style="width:163px;">Thermo-Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Waterbath</td>
			<td>Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Temperature-controlled shaker</td>
			<td>New Brunswick Scientific</td>
			<td style="width:119px;">C25KC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Sorvall Legend XTR centrifuge</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75004521</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Swinging Bucket Centrifuge Rotor</td>
			<td>Thermo-Scientific</td>
			<td style="width:119px;">75003607</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge LB-M</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW28 Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">342204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SW41 Ti Ultracentrifuge Rotor</td>
			<td>Beckman-Coulter</td>
			<td style="width:119px;">331336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Western blot</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">IRDye 680 Goat Anti-Rabbit IgG</td>
			<td style="width:163px;">LI-COR</td>
			<td style="width:119px;">926-68071</td>
			<td>dilution 1:15000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">IRDye&#160; 800CW Goat Anti-Mouse IgG</td>
			<td style="width:163px;">LI-COR</td>
			<td style="width:119px;">926-32210</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">NuPAGE&#174; Novex&#174; 12% Bis-Tris gels</td>
			<td style="width:163px;">Invitrogen</td>
			<td style="width:119px;">NP0341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">primary antibodies for PROTOCOL #1</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Erg6p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Dpm1p</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab113686</td>
			<td>4 &#956;g/ml</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Por1p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:5000</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:353px;">Pma1p</td>
			<td style="width:163px;">gift from Dr. G. Daum</td>
			<td style="width:119px;">Graz University of Technology, Austria</td>
			<td>dilution 1:10000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Vma1p (anti-ATP6V1A)</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab113745</td>
			<td>0.5 &#956;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;"></td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">primary antibodies for PROTOCOL #2</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">perilipin 2 (anti-ADFP)</td>
			<td style="width:163px;">Abcam</td>
			<td style="width:119px;">ab52355</td>
			<td>2 &#956;g/ml</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">calnexin</td>
			<td style="width:163px;">Cell Signaling technology</td>
			<td style="width:119px;">2679</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">GM130</td>
			<td style="width:163px;">Biorbyt</td>
			<td style="width:119px;">orb40533</td>
			<td>dilution 1:25</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:353px;">COX IV</td>
			<td style="width:163px;">Cell Signaling technology</td>
			<td style="width:119px;">4850</td>
			<td>dilution 1:1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">MEK1</td>
			<td style="width:163px;">Biorbyt</td>
			<td style="width:119px;">orb38775</td>
			<td>dilution 1:50</td>
		</tr>
	</tbody>
</table>

isolation of cellular lipid droplets: two purification techniques starting from yeast cells and human placentas

Lipid damlacıkları en ökaryotik ve prokaryotik hücrelerde bazı bulunabilir dinamik organellerdir. Yapısal olarak, damlacıkları, bir fosfolipid tek tabaka ile çevrilidir nötr lipitlerin bir çekirdekten oluşur. Damlacıkların hücresel rolleri belirlenmesinde en yararlı tekniklerden biri damlacıkları ile birlikte izole edilebilir bağlı proteinler, proteomik tanımlanması olmuştur. Fizyon maya ve insan plasental villöz hücreleri: Burada, iki yöntem, iki geniş ökaryotlardan lipid damlacıkları ve bunların bağlı proteinleri izole etmek için tarif edilmiştir. Her iki teknik farklılıklar olmasına rağmen, ana yöntem - yoğunluk gradyan santrifüj - hem hazırlıkları tarafından paylaşılır. Bu sunulan damlacık izolasyon teknikleri geniş uygulama gösterilmektedir. İlk protokolde, maya hücreleri, hücre duvarlarının enzimatik sindirme ile spheroplastlar dönüştürülür. Oluşan sferoplastlar sonra Gently bir gevşek homojenizatör içinde eritildi. Ficoll yoğunluk gradyanı sağlamak için lizat ilave edilir ve karışım, üç kez santrifüj edilir. Ilk dönüşü sonra, lipid damlacıkları, endoplazmik retikulum (ER), plazma zarı ve vakuoller ile birlikte santrifüj tüplerine beyaz renkli bir kayan tabakaya lokalizedir. Daha sonraki iki spin bu diğer üç organellere kaldırmak için kullanılır. Sonuç olarak sadece damlacıkları ve bağlanmış proteinler sahip bir tabakadır. İkinci protokolde, plasenta villöz hücreler tripsin ve DNase I Hücreler, gevşek homojenleştiricide homojenize edilir ile enzimatik sindirme ile insan plasentası terimi izole edilmiştir. Düşük hız ve orta hızlı santrifüj adımları kırılmamış hücreleri, hücresel atıkları, çekirdek ve mitokondri kaldırmak için kullanılır. Sakaroz bir yoğunluk gradyanı sağlamak için homojenat ilave edilir ve karışım, diğer cellula gelen lipid damlacıkları ayırmak için santrifüj edilirr kesirler. Her iki protokolde de lipid damlacıkları saflığı, Western Blot analizi ile teyit edilir. Her iki preparatlarıyla gelen damlacık fraksiyonlar sonraki proteomik ve lipidomic analiz için uygundur.

Beklendiği gibi yoğunluk gradyan santrifüj çalıştıysanız, yüzen tabaka lipid damlacıkları içermelidir ve yüksek hızda spin ilerlemesi boyunca diğer organellere tükenmiş olabilir. Protokol 1 için, Western blot işaretleyici lipid damlacıkları (Erg6p) antikorları ve maya, ER (Dpm1p), mitokondri (Por1p) lipid damlaları ile etkileşim için tespit edilmiştir organel ile yapıldı, plazma membranı (Pma1p) ve vakuol (Vma1p). Her üç spin yüzer...

Watch this Scientific Journal Video about Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas at JoVE.com

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

: 1) hücresel lipid damlacıkları izole maya hücreleri ve 2) insan plasenta sunulmuştur için iki teknikleri. Her iki prosedürlerinin merkezinde damlacıkları ihtiva eden elde edilen yüzer tabaka, hali hazırda, göz ile görselleştirilmiştir ekstre edilmiş ve saflık için Western Blot analizi ile tayin edilebilir yoğunluk gradyan santrifüj vardır.

Hücresel Lipid damlacıklar İzolasyonu: Maya Hücreleri ve insan plasentası itibaren iki Arıtma Teknikleri

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

isolation-cellular-lipid-droplets-two-purification-techniques

Research

JoVE Journal

Bioengineering

19.5K Görüntüleme.  University of Tennessee.  : 1) hücresel lipid damlacıkları izole maya hücreleri ve 2) insan plasenta sunulmuştur için iki teknikleri. Her iki prosedürlerinin merkezinde damlacıkları ihtiva eden elde edilen yüzer tabaka, hali hazırda, göz ile görselleştirilmiştir ekstre edilmiş ve saflık için Western Blot analizi ile tayin edilebilir yoğunluk gradyan santrifüj vardır. 

Video: Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

İnsan Hücre Kültürü Döner Hücre Kültür Sistemleri: Bir Model Olarak İnsan trofoblast Hücreleri

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Biyomühendislik

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

Hücresel Lipid Ekstraksiyon Hedefli Kararlı İzotop Seyreltme Sıvı Kromatografi-Kütle Spektrometresi Analizi

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

Geliştirme, Genişleme ve<em&gt; In vivo</em&gt; İnsan Embriyonik Kök Hücreleri (hESC) den İnsan NK Hücrelerinin İzleme ve ve Bağlı Pluripotent Kök Hücre (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Mikroakışkan jeti kullanarak çift katlı lipid Vesikül Üretimi

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Isolation of Primary Human Colon Tumor Cells from Surgical Tissues and Culturing Them Directly on Soft Elastic Substrates for Traction Cytometry

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors1, 2. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro. Here, an optimized protocol is developed that describes the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses using free open access software. In addition, immunofluorescence microscopy on different stiffness substrates provides useful information about primary cell morphology, cytoskeleton organization and vinculin containing focal adhesions as a function of substrate rigidity.

A protocol is described to extract primary human cells from surgical colon tumor and normal tissues. The isolated cells are then cultured on soft elastic substrates (polyacrylamide hydrogels) functionalized by an extracellular matrix protein, and embedded with fluorescent microbeads. Traction cytometry is performed to assess cellular contractile stresses.

Cerrahi Dokular İlköğretim İnsan Kolon Tümör Hücreleri İzolasyon ve Çekiş Sitometrinin Yumuşak Elastik Yüzeyler Doğrudan onları Kültürleme

Cancer cells respond to matrix mechanical stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Etanol ile lignoselüloz biyo dönüşümünü için Sağlam Pentoz-fermente Maya Evrimi Teknikleri

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Yalıtım insan alveoler kapaklarini ve Osteojenik etkinlik kapaklarini türetilmiş hücre D vitamini etkileri Mezenkimal kök hücrelerin

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Falcarindiol tarafından insan mezenkimal kök hücre içeren yeni Lipid kaplı nano tanecikleri alımını

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Erişkin İnsan Dermal Fibroblastlarının Abdominal Deriden İzolasyon ve İndüklenen Pluripotent Kök Hücrelerin Indüklenen Alacalı Kök Hücrelerin Indükleyici Olmayan Yöntemle Üretilmesi

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

İnsan kaynaklı Pluripotent Kök Hücre Türevli Kardiyomiyositlerde Tek Hücreli Optik Eylem Potansiyeli Ölçümü

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...