This work was supported by the Seed Grant Award from California Northstate University College of Pharmacy (to AMN). The authors would like to thank California Northstate University College of Pharmacy for covering the publication cost of this article, and George Talbott for his help in editing this manuscript.

CACO-2 hücrelerinin 1. Bakım <ol><li> Düzenli doku kültürü yemekleri kullanma <ol><li> (FBS, 37 ° C su banyosu içinde şişe yerleştirerek bir dondurulmuş şişesinden alınan Caco-2 hücreleri çözülme ve hemen önceden ısıtılmış büyüme ortamı (% 15 fetal bovin serumu, 10 ml içeren 10 cm&#39;lik bir doku kültürü kabı eklemek ) Dulbecco Modifiye Edilmiş Eagle Ortamı (DMEM)) &#39;de. <ol><li> Caco-2 hücreleri% 50-70 kaynaşmaya ulaştığı zaman, onları 1 bölünmüş: müstakil kadar 37 ° C&#39;de% 0.05 tripsin / 0.53 mM etilendiamintetraasetik asit (EDTA), 3 ml ile hücrelerin inkübe edilmesi ile 6 (15 dk) . Hücre topaklaşmayı önlemek yumuşak pipetleme hücreler karıştırmak için birkaç kez. Önceden ısıtılmış büyüme ortamı 10 ml içeren 10 cm&#39;lik bir doku kültürü kabı tripsinize edilmiş hücre 0.5 ml ilave edilir. </li></ol></li><li> Yavaşça bir sola ve sağa yön ardından ileri ve geri yönde yemeklere birkaç kez sallayın. Yemekler dönen kaçınıngibi eşit olmayan bir hücre dispersiyonu ile sonuçlanabilir. </li><li> % 5 CO 2 ile birlikte 37 ° C inkübatör düz bir yüzeye yerleştirin yemekleri. Eğimli yüzeyler de eşit olmayan bir hücre dispersiyonu ile sonuçlanabilir. </li><li> Inkübasyon başladıktan sonra büyüme Ortam bir gün değiştirin. </li><li> Hücreleri izlemek ve izdiham ulaşmak günü kaydedin. Hücreler bölünür günden itibaren izdiham ulaşmak için yaklaşık 1 hafta sürer. </li><li> Hücreler izdiham ulaşmadan haftada iki kez büyüme ortamı değiştirin. Hücreler izdiham ulaştığınızda, her gün büyüme ortamı değiştirmek. Hücreler 7-günler sonrası konfluent olduktan sonra, günlük büyüme ortamı değiştirmek. Not: 13-günler sonrası konfluent (Şekil 1) olduğunda hücreler deneyler için hazırız. Hücreler sonunda lipoproteinlerin üreten daha az etkili hale gelecektir yana, 8 sonrası birleşik 13-17-gün arasında olan hücreleri kullanır. Deney yapmak için nasıl adım 3&#39;e gidin.</li></ol></li><li> Geçirgen zar sistemi kullanılarak <ol><li> Aşama 1.1.1.1 tarif edildiği gibi geçirgen zar ekinde Caco-2 hücreleri, tohum. Yukarıda. Onların hücre büyümesi (iyileşme dönemi) normalde yavaş çünkü dondurulmuş şişesinden alınan hücreler kullanılarak kaçının. Kısaca, önceden ısıtılmış büyüme ortamı 10 ml içeren apikal (üst bölmesi) tripsinize edilmiş hücre 0.5 ml ilave ediniz. Ayrıca, bazolateral bölme (alt bölmesi) önceden ısıtılmış büyüme ortamı 10 ml. Not: En iyi uygulama için, bazolateral odasına ilk ve daha sonra apikal odasına büyüme ortamı ekleyin. Eski medya çıkartırken, apikal medya önünde bazolateral ortamı çıkarın. Bu membran rüptürü olabilir polikarbonat membran alay kaçının. </li><li> Nedeniyle polikarbonat zardan kötü hücre görünürlüğe, benzer yoğunlukta düzenli bir doku kültürü çanak Caco-2 hücreleri tohum ve hücre izdiham yargılamak için bu yemeğin kullanın. </li><li> S izleyinTeps&#39;in 1.1.2-1.1.6, yukarıda. </li></ol></li></ol> 2. Gene overekspresyonu <ol><li> Normal transfeksiyon yaklaşımı kullanarak <ol><li> Yukarıda 1.1.1.1.-1.1.4 de tarif edildiği gibi bir doku kültürü tabağına Caco-2 hücreleri, tohum. Not: hücreler yaklaşık% 40-50 konfluent olduğunda, transfekte hazırdır. </li><li> Mikrosantrifüj tüpü içinde düşük serum ortamında 472 ul transfeksiyon reaktifi 67 ul ekle. Tüp duvar ile sulandırılmamış transfeksiyon reaktif temasından kaçının. </li><li> PLL3.7 23 ug transfeksiyon reaktifi / düşük serum ortamında karışımına, yeşil flüoresan protein (EGFP) 9 Geliştirilmiş ekleyin. </li><li> Oda sıcaklığında 20 dakika süreyle inkübe karışımı. </li><li> DMEM içinde% 10 FBS 8 ml Caco-2 hücreleri, büyüme ortamı değiştirin. </li><li> Damla damla çanak DNA / transfeksiyon reaktif / azaltılmış serum medya karışımı ekleyin. </li><li> Yavaşça ileri ve ba yemekleri birkaç kez sallayınBir sola ve sağa yönde takip ckward yön. </li><li> 37 ° C inkübatör çanak yerleştirin. </li><li> Kuluçka başladıktan sonra büyüme ortamı 10 ml her gün bir çanak ortamı değiştirin. </li><li> Gen ifadesini izleyin. <ol><li> 4 &#39;, 6-diamidino-2-fenilindol (DAPI) ile boyanmamış olan gen ekspresyonunu izlemek. Floresan mikroskobu kullanılarak, yeşil hücrelerin yüzde belirler. yüzde transfeksiyon verimini gösterir. </li><li> DAPI boyama ile gen ifadesini izleyin. <ol><li> Fosfat tamponlu salin (PBS) ile üç kez hücreler yıkanır. </li><li> PBS (oda sıcaklığında 10 dakika inkübasyon) içinde% 4 formaldehit, 10 ml ekleyerek hücreleri saptamak. </li><li> PBS üç kez hücreleri yıkayın. </li><li> 500 DAPI,% 1 BSA,% 0.01 digitonin: 1 içeren PBS 8 ml ile oda sıcaklığında 15 dakika süreyle karanlıkta inkübe hücreleri. </li><li> PBS üç kez hücreleri yıkayın. </li><li> U, bir flüoresan mikroskop şarkı, mavi (Şekil 2 üst panel) ki bu yeşil / mavi göreli hücre sayısını belirler. hesaplanan yüzde transfeksiyon verimini gösterir. </li></ol></li></ol></li></ol></li><li> Lentivirüs aşırı sistemi 10 kullanılarak <ol><li> 15 cm&#39;lik bir doku kültürü çanağı içinde Tohum, insan embriyonik böbrek (HEK) 293T hücreleri (DMEM içinde% 10 FBS büyüme ortamı olarak). Not: hücreler yaklaşık% 60-70 konfluent olduğunda, transfekte hazırdır. </li><li> Mikrosantrifüj tüpü 11 düşük serum ortamı 1133 ul transfeksiyon reaktifi 162 ul ekle. Tüp duvar ile sulandırılmamış transfeksiyon reaktif temasından kaçının. </li><li> Ekle 24 pLL3.7 eGFP (ya da başka bir yapı) ug, 15.6 REV karşılık elemanının ug (RRE), REV 6.0 ug, ve transfeksiyon reaktifi / r içine veziküler stomatit virüsü glikoprotein 8.4 ug (VSVG)educed serum ortamında karışımı ile gerçekleştirilmektedir. </li><li> Oda sıcaklığında 20 dakika süreyle inkübe karışımı. </li><li> DMEM içinde% 10 FBS ve 16 ml büyüme ortamı değiştirin. </li><li> Damla damla çanak DNA / transfeksiyon reaktif / azaltılmış serum medya karışımı ekleyin. </li><li> Yavaşça bir sola ve sağa yön ardından ileri ve geri yönde yemeklere birkaç kez sallayın. Bu noktada müstakil HEK293T hücrelerinin bazılarını görmek için normaldir. </li><li> Inkübatör çanak yerleştirin. </li><li> Kuluçkalamadan 24 saat sonra, büyüme ortamı, 25 ml çanak ortamı değiştirin. </li><li> Ertesi gün, lentivirüs (birinci toplama) içeren büyüme ortamı toplar. </li><li> Adımı yineleyin 2.2.10 fazla lentivirüs (ikinci toplama) toplamak için. </li><li> (5 dakika boyunca 2.500 xg) birinci ve ikinci toplama havuzu ve santrifüj hücre enkaz kaldırmak. </li><li> Tek kullanımlık şişe üstü filtre (0.45 mikron gözenek) ile toplama Filtre veRT (JA-20 rotor) 2 saat süre ile 31.400 x g&#39;de santrifüje toplama virüsü konsantre edilir. Pelet bulunduğu santrifüj tüpünün dibine işaretleyin. Süpernatant tortusundan ayırma, ve yaklaşık 15 dakika boyunca oda sıcaklığında ters tüpü terk. </li><li> 1X PBS, 100 ul virüs içeren pelletini. Kabarcıkları kaçının. </li><li> -80 ° C &#39;de konsantre edilir virüs saklayın. Donma-çözülme döngüleri kaçının. </li><li> Titrasyon ile, Caco-2 hücreleri nakletmek için gerekli virüs optimal miktarının belirlenmesi. Maliyet tasarrufu için, 24-çukurlu plaka (iyi büyüme ortamı / 0.3 ml) kullanılarak titrasyonu için 10 cm&#39;lik bir doku kültürü çanağı kullanın. <ol><li> Konsantre edilmiş virüs artan miktarlarda ekleyin (0, 1, 2, 5, 10, 25, ve 50 ul / kuyucuk)% 40-70 konfluent Caco-2 hücreleri için polibren (nihai konsantrasyon = 5 ug / ml) ile takviye edilmiştir. 1.1.2 tarif edildiği gibi hafifçe 24 oyuklu plaka çalkalayın. </li><li> Onların gen ifadesini Monitör (Adım 2.1.10.) (Şekil 2 alt4 paneller). </li></ol></li><li> Transgen ifadesi genellikle sürekli olduğundan, gelecek deneyler için transdüse hücreleri korumak ve sürekli olarak gen ifadesini izlemek (Adım 2.1.10.). </li></ol></li></ol> 3. Lipoprotein salgılanmasını uyararak <ol><li> Düzenli doku kültürü çanak kullanma <ol><li> Oleik asit, 50 mg, lesitin, 40 mg, ve sodyum taurokolat ve 48 mg karıştırılarak bir 100X yağ karışım hazırlayın. (8 yemekler için yeterli) PBS ile 900 ul hacim kazandırın. Kuvvetli bir şekilde lipit karışımı karıştırın. </li><li> Büyüme ortamı 89.1 ml 100X lipit karışımı 900 ul ekleyin. Mix ve lipid içeren medya filtre. oleik asit, lesitin, ve sodyum taurokolat ve nihai konsantrasyonlar (1X) 2.0 mM, 1.36 mM ve sırası ile 1.0 mM vardır. </li><li> Hücrelere lipid içeren ortam, 10 ml ilave edilir. </li><li> 37 4 saat boyunca lipit-içeren ortam ile inkübe hücreleriC inkübatör °. </li><li> PBS üç kez hücreleri yıkayın. </li><li> Lipoprotein salgılanmasını toplamak için hücrelerin büyüme ortamı 10 ml ilave edilir. </li><li> 37 ° C kuluçka makinesi içinde 2 saat süreyle inkübe edin. </li><li> Lipoprotein içeren medya toplayın. </li></ol></li><li> Geçirgen zar sistemi kullanılarak <ol><li> Adımları 3.1.1-3.1.2 lipit karışımı hazırlamak için. </li><li> Apikal bölmeye lipid içeren ortam 10 ml ve bazolateral bölmeye büyüme ortamı 10 ml ilave edilir. </li><li> 37 ° C kuluçka makinesi içinde 4 saat süreyle inkübe edin. Bazolateral bölme lipoprotein ihtiva eden ortam toplayın. </li></ol></li></ol> 4. lipoprotein İzolasyon (Şekil 3) <ol><li> Santrifüj (5 dakika boyunca 2.000 xg) lipoprotein içeren medyanın hücre enkaz kaldırmak. </li><li> 50 ml&#39;lik bir tüp içine ortamı süzün. </li><li> Ortam, sodyum klorür 5.95 g ekleyin. </li><li> Sa Eğermple TEM ile analiz edilecektir, lipoproteinlerin bütünlüğünü korumak için 1 proteaz önleyici kokteyl tablet ekleyin. </li><li> Su ile 23 ml hacmi (1.2 g / ml NaCI yoğunluk çözeltisi) getirin. </li><li> Tamamen çözünmüş çözülür. </li><li> Polikarbonat ultrasantrifüjdeki tüp içine çözeltinin Tümünü süzün. </li><li> Yavaşça, 0.5 ml, su (1.0 ug / ml yoğunlukta), 1.2 ug / ml yoğunlukta çözelti kaplar. </li><li> Hacimce ağırlıkça değil tüp dengeleyin. </li><li> Yavaşça T865 rotor tüpler yükleyin. </li><li> 4 ° C&#39;de 24 saat boyunca 429.460 x g&#39;de ultrasantrifüjdeki örnekleri dönerler. </li><li> Hemen Nazik pipetleme üst 0.5 ml solüsyon izole. Mümkün olduğunca hareketsiz tüp tutun. </li></ol> 5. TEM analizi <ol><li> (A / h) ile pH 6,0&#39;a ayarlanır% 2 fosfotungstik asit 5 ml hazırlayın. </li><li> (: 0.2 mikron gözenek boyutu) bir şırınga filtresi ile% 2 fosfotungstik asit Filtre. </li><li> Steril bir çanak lipoprotein numunenin 20 ul bırakın. </li><li> Bir sonraki aynı yemeğin üzerine örnek süzülmüş% 2 fosfotungstik asit 20 ul bırakın. </li><li> Yavaşça donuk yan numune üzerinde oturan bir EM ızgara bırakın. </li><li> 1 dakika süre ile, oda sıcaklığında inkübe edilir. </li><li> Yavaşça ızgara örneği kaldırmak için bir filtre kağıdına EM ızgara tarafı dokunun. </li><li> Yavaşça donuk tarafı% 2 fosfotungstik asit dayayarak EM ızgara bırakın. </li><li> 1 dakika süre ile, oda sıcaklığında inkübe edilir. </li><li> Yavaşça ızgara fosfotungstik asit ayrılması için bir filtre kağıdı üzerinde EM ızgara yan dokunun. </li><li> Bir TEM kullanarak, lipoproteinler (Şekil 4) görüntüleri yakalamak. </li></ol>

<ol>
	<li>Drover, V. A., 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=CD36+deficiency+impairs+intestinal+lipid+secretion+and+clearance+of+chylomicrons+from+the+blood.">CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood.</a> J Clin Invest. 115 (5), 1290-1297 (2005).</li><li>Nauli, A. M., 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=CD36+is+important+for+chylomicron+formation+and+secretion+and+may+mediate+cholesterol+uptake+in+the+proximal+intestine.">CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine.</a> Gastroenterology. 131 (4), 1197-1207 (2006).</li><li>Nauli, A. M., Zheng, S., Yang, Q., Li, R., Jandacek, R., Tso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+release+is+not+associated+with+chylomicron+formation.">Intestinal alkaline phosphatase release is not associated with chylomicron formation.</a> Am J Physiol Gastrointest Liver Physiol. 284 (4), G583-G587 (2003).</li><li>Lo, C. -. M., 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=Why+does+the+gut+choose+apolipoprotein+B48+but+not+B100+for+chylomicron+formation.">Why does the gut choose apolipoprotein B48 but not B100 for chylomicron formation.</a> Am J Physiol Gastrointest Liver Physiol. 294 (1), G344-G352 (2008).</li><li>Jandacek, R. J., Heubi, J. E., Tso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel,+noninvasive+method+for+the+measurement+of+intestinal+fat+absorption.">A novel, noninvasive method for the measurement of intestinal fat absorption.</a> Gastroenterology. 127 (1), 139-144 (2004).</li><li>Nauli, A. M., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+transport+as+a+potential+determinant+of+drug+bioavailability.">Intestinal transport as a potential determinant of drug bioavailability.</a> Curr Clin Pharmacol. 8 (3), 247-255 (2013).</li><li>Tso, P., Nauli, A., Lo, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterocyte+fatty+acid+uptake+and+intestinal+fatty+acid-binding+protein.">Enterocyte fatty acid uptake and intestinal fatty acid-binding protein.</a> Biochem Soc Trans. 32 (Pt 1), 75-78 (2004).</li><li>Nauli, A. M., Sun, Y., Whittimore, J. D., Atyia, S., Krishnaswamy, G., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chylomicrons+produced+by+Caco-2+cells+contained+ApoB-48+with+diameter+of+80-200+nm.">Chylomicrons produced by Caco-2 cells contained ApoB-48 with diameter of 80-200 nm.</a> Physiol Rep. 2 (6), (2014).</li><li>Rubinson, D. A., 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=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33 (3), 401-406 (2003).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Pottekat, A., 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=Insulin+biosynthetic+interaction+network+component,+TMEM24,+facilitates+insulin+reserve+pool+release.">Insulin biosynthetic interaction network component, TMEM24, facilitates insulin reserve pool release.</a> Cell Rep. 4 (5), 921-930 (2013).</li><li>Van Greevenbroek, M. M., van Meer, G., Erkelens, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+saturated,+mono-,+and+polyunsaturated+fatty+acids+on+the+secretion+of+apo+B+containing+lipoproteins+by+Caco-2+cells.">Effects of saturated, mono-, and polyunsaturated fatty acids on the secretion of apo B containing lipoproteins by Caco-2 cells.</a> Atherosclerosis. 21 (1), 139-150 (1996).</li><li>Levy, E., Yotov, W., Seidman, E. G., Garofalo, C., Delvin, E., M&#233;nard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caco-2+cells+and+human+fetal+colon:+a+comparative+analysis+of+their+lipid+transport.">Caco-2 cells and human fetal colon: a comparative analysis of their lipid transport.</a> Biochim Biophys Acta. 1439 (3), 353-362 (1999).</li><li>Luchoomun, J., Hussain, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+secretion+of+chylomicrons+by+differentiated+Caco-2+cells.+Nascent+triglycerides+and+preformed+phospholipids+are+preferentially+used+for+lipoprotein+assembly.">Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly.</a> J Biol Chem. 274 (28), 19565-19572 (1999).</li></ol>

The author has nothing to disclose.

Bu yazıda, kullanılabilecek iki sistem Caco-2 hücreleri, yani düzenli doku kültürü çanak ve geçirgen zar açıklanmıştır korumak için. geçirgen zar sistemi kullanmanın faydaları apikal ayrılmasını ve bazolateral bölmeleri ve lipid karışımı inkübe ve aynı zamanda lipoprotein salgılanmasını toplamak için yeteneği vardır. Ancak, geçirgen zar ekler pahalıdır, ve polikarbonat membran iyi bir hücre görünürlüğü izin vermez. Bu in vitro modelin avantajlarından biri genetik man...

4 - diyet yağ ve lipid içinde çözünür ilaçlar ve vitaminler intestinal çalışmaları lenf fistül MODEL 1 kullanılarak in vivo olarak gerçekleştirilebilir. Ancak, ilgili cerrahi teknikler sadece zor değil, aynı zamanda pahalı değil. In vivo kullanılabilmektedir dışkı analizi dayalı yaklaşımlar da, mide-barsak sisteminde 2,5 yüzdesi alımını belirlemek için temel olarak kullanılmaktadır. Bu yazıda anlatılan in vitro model daha düşük maliyetli olduğunu ve ilgili teknikler tartışmalı az zorlayıcı bulunmaktadır. Genetik modifikasyonlar çalışmaları da zaman alıcı, bu in vitro bir model kullanılarak gerçekleştirilmiştir zaman daha ekonomik ve daha az bulunmaktadır. Enterositler tarafından alınır lipit çözünür malzeme lipoproteinler 6,7 içine paketlenmiş olduğundan, lipoproteinlerin üretmek için bu in vitro modelin etkinliği çok önemlidir. iki ana intestinal lipoproteinler kilomikronlar, çok düşük yoğunluklu lipoproteinler (VLDL) vardır. Lipidler mide-bağırsak lümeninde bol mevcut olduğunda çapı 80 nm ya da daha fazlası ile lipoproteinler olarak tanımlanan kilomikronlar, küçük bağırsak tarafından sıkı bir şekilde üretilir. Onlar büyük lipoproteinler olduğundan, şilomikronlar makul en verimli lipit taşıyıcıları vardır. Şilomikron 8 üretebilen bu in vitro modeli, diyet yağ emme, gut lipid çözünür vitamini emme ve oral lipofilik ilaç biyo-incelemek için kullanılabilir. lipoprotein fraksiyonuna lipid çözünür moleküller, vitaminler ya da ilaçların bulunması, ince bağırsakta kendi emilme bir göstergesidir. Daha önce ele alındığı gibi, bu model, oral lipofilik ilaç biyo-6 geliştirmek için de kullanılabilir. Bu kağıt Caco-2 hücreleri geçirgen zar veya normal doku kültürü yemekleri, nasıl lipit mi muhafaza edilmelidir açıklanırLipoprotein üretimini stimüle etmek için soğutun lentivirüs sentezleme sisteminin etkin aşırı ekspresyonunu gerçekleştirmek için kullanılabilir nasıl hazırlanmalı ve izole lipoproteinler kadar analiz edilmelidir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>VWR</td>
			<td>16750-112</td>
			<td>Pre-warm the growth media in individual tissue culture dish before adding cells</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Fisher</td>
			<td>3600511</td>
			<td>We do not heat inactivate our serum</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-660</td>
			<td>Cells can be washed with PBS prior to trypsin treatment&#160;</td>
		</tr>
		<tr>
			<td>Permeable membrane system</td>
			<td>Fisher</td>
			<td>7200173</td>
			<td>10-cm dish, 3-mm pore size, polycarbonate membrane</td>
		</tr>
		<tr>
			<td>10 cm dish</td>
			<td>Fisher</td>
			<td>08-772-E</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr>
			<td>15 cm dish</td>
			<td>Fisher</td>
			<td>08-772-24</td>
			<td>Tissue culture dish</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Fisher</td>
			<td>12565163</td>
			<td>Tissue culture plate</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent</td>
			<td>Fisher</td>
			<td>PRE2691</td>
			<td>Can be substituted with other transfection reagent</td>
		</tr>
		<tr>
			<td>Reduced serum media</td>
			<td>Invitrogen</td>
			<td>11058021</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>pLL3.7 eGFP</td>
			<td>Addgene</td>
			<td>11795</td>
			<td><a href="https://www.addgene.org/11795/">https://www.addgene.org/11795/</a></td>
		</tr>
		<tr>
			<td>Bottle-top filter</td>
			<td>Fisher</td>
			<td>9761120</td>
			<td>0.45 mm pore</td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Fisher</td>
			<td>NC9840454</td>
			<td>10 mg/mL</td>
		</tr>
		<tr>
			<td>Oleic acid</td>
			<td>Sigma</td>
			<td>01383-5G</td>
			<td>Prevent freeze-thaw cycle</td>
		</tr>
		<tr>
			<td>Lecithin&#160;</td>
			<td>Fisher</td>
			<td>IC10214625</td>
			<td>Egg lecithin&#160;</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Fisher</td>
			<td>NC9620276</td>
			<td>Product discontinued; alternative catalog number: 50-121-7956</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail tablet (EDTA-free)</td>
			<td>Fisher</td>
			<td>5892791001</td>
			<td>Used mainly for samples that need TEM analysis</td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuge tube</td>
			<td>Fisher</td>
			<td>NC9696153</td>
			<td>Reusing it multiple times will collapse the tube</td>
		</tr>
		<tr>
			<td>Lid for ultracentrifuge tube</td>
			<td>Fisher</td>
			<td>NC9796914</td>
			<td>A tool is required to remove the tube/lid from rotor</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Fisher</td>
			<td>50-949-261</td>
			<td>Disposable</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Fisher</td>
			<td>09-719C</td>
			<td>Pore size = 0.2 mm; nylon</td>
		</tr>
		<tr>
			<td>Phosphotungstic acid</td>
			<td>Fisher</td>
			<td>AC208310250</td>
			<td>For preparing 2% phosphotungstic acid, pH 6.0</td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Fisher</td>
			<td>50-238-62</td>
			<td>Extra fine and strong tips</td>
		</tr>
		<tr>
			<td>Formvar/carbon grid</td>
			<td>Fisher</td>
			<td>50-260-34</td>
			<td>Formvar/carbon film square grid 400 Copper</td>
		</tr>
	</tbody>
</table>

using caco-2 cells to study lipid transport by the intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

1 görüntüler, normal 13-gün sonrası birleşik Caco-2 hücreleri Şekil. Kubbe-şekilli yapılar ve hücre içi lipid damlacıkları görünümü farklılaşmış Caco-2 hücreleri için karakteristiktir. Caco-2 hücreleri tohumlama sırasında eşit dağılmış değil, onlar kümelenir ve yemeğin belirli alanlarda sarmak olacaktır; ve herhangi bir hücrelerde olmadan çanak birkaç alanları olacaktır. Swirling ve eğimli bir yüzeye çanak yerleştirilmesi kaçınılmalıdır....

Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Bağırsak tarafından Lipit Transport Eğitim için Caco-2 hücreleri kullanarak

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

using-caco-2-cells-to-study-lipid-transport-by-the-intestine

Research

JoVE Journal

Biology

18.6K Görüntüleme.  California Northstate University. Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Video: Using Caco-2 Cells to Study Lipid Transport by the Intestine

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Kertenkeleler Hareket Hız ve Yön Visual Hassasiyet Test

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Biyoloji

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Bilgisayar oluşturuldu Hayvan Modeli Uyaranlar

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Na, K ve H, K-ATPaz bölgesindeki tarafından Katyon Taşıma Ölçüm<em&gt; Xenopus</emAtomik Absorpsiyon Spektrofotometresi ile&gt; Oosit: Radyoizotop Tahliller Alternatif Bir

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

Yöntem 3D Yetiştirilen Yerli Barsak ve Caco-2 Hücreler Epitel Ulaşım Protein Fonksiyonu ve Anlatım Eğitim için

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Enteroendokrin Hücre Gut Hormon salgısını Çalışması ve Canlı Hücre Görüntüleme için Amaçlanan Kemirgen Küçük Bağırsak Karışık İlköğretim Kültürler

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Altın Nanopartiküller ve Lipid Membranları Arasındaki Isı Transferini İzlemek için Bağlı Bilayer Lipid Membranları

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

Aort Kapak In Vitro Kalsifikasyonu Çalışmak için Fare Interstisyel Kapak Hücrelerinin İzolasyonu

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Gıda Demir Biyoyararlanımının Ölçümü için Caco-2 Hücre Biyotahlili

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from&#160;lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.

Matris Destekli Lazer Desorpsiyon/İyonizasyon Kütle Spektrometrisi Görüntüleme Kullanarak Drosophila Beyinde Hızlı Lipid Analizi için Örnek Hazırlama

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired ...

Memeli Hücrelerinde Çinko Taşıma Mekanizmasının FluoZin-3 Testi ile İncelenmesi

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Yazar Spotlight: ZnT1 İşlevselliği ile Hücresel Çinko Düzenlemesini Keşfetmek 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

İn Vitro Çinko Taşıma Testi Kullanılarak Memeli Çinko Taşıyıcılarının Karakterize Edilmesi

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...