Biz Astrosit kültürlerin cömert temini için Dr Stephen Robinson ve Ms Hanya Czerwińska (Monash Üniversitesi) müteşekkiriz.

1.. Kültür Hücreleri Hücre içi askorbat belirleyin <ol><li> Hücre kültürü ve hasat <ol><li> Standart kültür prosedürler 19-21,32,38 kullanılarak süspansiyon (örneğin insan eritrolösemi, K562) veya yapışık hücreleri (primer astrositlere) büyür. Not: hücreler, askorbat içeren askorbat ya da askorbat veya DHA ile 33,39 gibi kültür hücreleri yük sağlamak. </li></ol></li><li> Bir askorbat içeren hücresel özü oluşturun <ol><li> Fosfat tamponlu tuzlu su (PBS) ile yıkanmış ve süspansiyon buz gibi soğuk hücre Permeabilization Tamponu [CPB 450 ul ile yapışan hücreler bir uygun sayıda inkübe et; PBS içinde% 0.1 (ağ / hac) bir saponin; 4 ° C]. Not: deneysel olarak son kullanıcı tarafından (aşağıya bakınız) deneyinin lineer aralığında bir absorbans değeri elde etmek için gerekli olan hücre sayısını belirler. Not: doku ekstreleri de (daha fazla bilgi için Tartışma) kullanılabilir. </li><li> Agita10 dakika boyunca buz üzerinde te ayrıntılı hücreleri hücre liziz sağlamak. </li><li> Soğutulmuş bir mikrosantrifüj (4 ° C) 5 dakika için 16,000 x g&#39;de ham lizat santrifüj ile hücre artıklarının çıkarılması ile berraklaştırılmış askorbat ihtiva eden hücre içi özü oluşturun. </li><li> Her bir numune dikkatli bir şekilde 4 x 100 ul alikotları kaldırmak ve tek ya da 25 ul / oyuk 45.5 U PBS (&quot;AO-&quot;), 25 ul / ya da ihtiva eden bir 96-gözenekli düz tabanlı plaka içindeki kuyu yatay dizisine eklemek / ml PBS (&quot;+ AO&quot;) L-askorbat oksidaz-(AO) stok çözeltisi. Not: Bu (aşağıya bakınız) standartlarının hazırlanması paralel olarak yapılmalıdır. </li></ol></li><li> Askorbat standart eğri yapı (her bir deney için yeniden inşa edilmesi gerekir) <ol><li> Buz soğukluğunda PBS içinde 10 mM askorbik asit, bir stok çözelti hazırlayın. Not: spektrofotometrik olarak 1 olan bir kuartz küvet kullanılarak askorbatın konsantrasyonu doğrulama265 nm&#39;de (yok olma katsayısı = 14.5 mM -1 cm-1) 40 cm yol uzunluğu. </li><li> Dikkatle 0 ve 20 mcM arasında askorbat standartların bir dizi hazırlamak. </li><li> Adım 1.2.4 paralel olarak, dikkatli bir şekilde, her bir standart 4 x 100 ul alikotları çıkarın ve PBS (&quot;AO-&quot;) ya da 25 ul / yuva ihtiva eden bir 96-gözenekli düz tabanlı plaka içindeki kuyu yatay dizisine eklemek PBS AO (&quot;+ AO&quot;) 45.5 U / ml stok solüsyonu. Not: Yukarıdaki askorbat standartların 100 ul askorbat 0-2 nmol içerir. Lütfen dikkat, AO amacı azalma ferricyanide olmayan askorbat redüktantların katkı için kontrol etmektir. </li></ol></li><li> Hücre içi askorbat belirlemek - Adım 1: seçici askorbat kaldırmak ve kantitatif ferrisiyanit ile askorbat okside <ol><li> Orbital folyo ile plaka kaplama ile karanlık bir ortamda 5 dakika boyunca oda sıcaklığında 550 rpm&#39;de 96 oyuklu plaka karıştırın. Not: Tüvey DHA&#39;ya &quot;+ AO&quot; kuyularda tüm askorbat okside gerekir. &quot;-AO&quot; kuyuları etkilenmemiş olmalıdır. </li><li> Tüm oyuklara PBS içinde (burada &quot;ferrisiyanür&quot; olarak anılacaktır) 3.5 mM potasyum ferrisiyanür 50 ul ekle. Nihai [ferrisiyanür] = 1 mM. Not: a multipipettor kullanın. </li><li> Orbital karanlıkta başka bir 5 dakika için oda sıcaklığında 550 rpm plaka karıştırın. Not: Bu adım okside askorbat 1 molekülü başına azaltılmış ferrisiyanit 2 molekülünün bir birleşme oranında askorbat ile ferrosiyanür için ferrisiyanür azalmaya neden olmalıdır. </li><li> Hemen% 50 (v / v) asetik asit ve% 30 (a / h) trikloroasetik asit (TCA) ihtiva eden bir yeni inşa çözeltisi 25 ul ekle. Not: a multipipettor kullanın. </li></ol></li><li> Hücre içi askorbat belirlemek - Adım 2: quantitate Ferrosiyanat miktarı 37 oluşmuş <ol><li> E ferrosiyanür belirlenmesi çözüm 37 100 ul ekleyiniyi ach. Not: bir çalışma çözeltisi kullanımdan hemen önce yapılmalıdır: 3 M Na-asetat (pH 6.0) içinde 2 ml; Buzlu asetik asit (~ 17.4 M asetik asit), 0.5 mi; 0.2 M sitrik asit ve 2 mi; 0.1 M asetik asit içinde 3.3 mM FeCl3 2 mi; 30 mM ferene-S 1 ml. Bu çalışma çözeltinin son hacmi 7.5 ml olmalıdır. </li><li> Orbital oda sıcaklığında 30 dakika boyunca 550 rpm&#39;de karanlıkta plaka karıştırın. </li><li> 593 nm&#39;de kuyuların absorbans değerleri oku (örneğin Fe (II) (ferene-S) 3 kompleksin absorbans maksimum). </li><li> (Adım 1.3 &#39;her bir örnek için -&#39; AO &#39;kuyuları ve daha sonra askorbat standart eğriden interpolating ilk olarak karşılık gelen&#39; + AO &#39;kuyular için bir 593 nm değerleri çıkarılarak milyon hücre başına nmol askorbat gibi hücre içi askorbat miktarını hesaplayın .) Standart eğri oluştururken, ascorb miktarına karşı her bir standart için, bu &quot;fark değeri&quot; arsaOyuk başına yedi. </li></ol></li></ol> Kültür Hücreler Askorbat efflux-2. Belirlenmesi <ol><li> Hücre kültürü ve hasat <ol><li> (Adım 1.1) yukarıdaki gibi süspansiyon veya yapışık hücreleri büyümek. Not: En iyi sonuçlar için süspansiyon ve yapışık hücreleri ile 24-iyi bir plat formatında aşağıda tahlil yürütmek. </li><li> Süspanse süspansiyon hücreleri ya da ya da, kalsiyum ve magnezyum olmaksızın, önceden ısıtılmış HEPES tamponlu salin çözeltisi 400 ul, yapışkan hücreler kaplamak ve 5 mM D-glukoz içeren, kuyularda (HBS / D, pH 7.3, 37 ° C) Bir 24-yuvalı plaka. Incelenecek her bir 24-çukurlu plaka üzerinde belirli bir hücre içermeyen çukurlara HBS / D aynı hacim. Not: ikinci (aşağıya bakınız) temel reaksiyon için hücre içermeyen kontroller olarak hizmet edecektir. </li></ol></li></ol> 3.. Çıkış Askorbat miktarı belirleyin <ol><li> Aşağıdaki stok çözeltileri önceden hazırlıklı olmalıdır: 120U / ml AO HBS / D (taze hazırlandı); 2.4 mM Ferene-S HBS / D; 120 uM FeCl3 ve HBS / D (daha fazla konsantre stok çözümleri hemen hazırlamak) 600 mcM Na-sitrat. </li><li> Ferrireduction reaksiyonu (her son hacim 600 ul olmalıdır) başlatmak için, daha önce HBS / D 400 ul ihtiva eden tek tek oyuklara reaktiflerin aşağıdaki miktarlar ekleyin: <ol><li> , Üç kopya halinde kuyu eşleştirilmiş için AO (120 U / ml) ya da HBS / D 50 ul ekle. Nihai AO konsantrasyon 10 U / ml olmalıdır. Not: Etiket olarak kuyu eşleştirilmiş &quot;- AO&quot; ve &quot;+ AO&quot;. </li><li> 37 ° C&#39;de 5 dakika boyunca hafif bir orbital karıştırma ile plakaları karıştırın </li><li> Tüm oyuklara 2.4 mM ferene-S 50 ul ekle. Nihai ferene-S konsantrasyonu, 200 uM olmalıdır. 37 ° C&#39;de 5 dakika boyunca, yukarıdaki gibi plakaları karıştırın </li><li> Taze hazırlanmış 120 uM ferrik sitrat 50 ul ekle. Nihai demir ve sitrat konsantrasyonları 10 mcM demir ve 50 mM sitrat olmalıdır. İyice karıştırın. </li></ol&gt; </li><li> Üç üçlü kontrol kaplan olarak, üstte uzanan orta aspire ve% 0.1 saponin içeren 600 ul HBS / D ekleyin. Not: Bu (aşağıya bakınız) paralel olarak yapılacak bir laktat dehidrojenaz (LDH) salım deneyi için &quot;% 100 hücre liziz&quot; kontrol grubu olarak hizmet edecektir. </li><li> 37 ° C&#39;de karanlıkta 60 dakika boyunca inkübe </li><li> Askorbat-akış deneyinde sonunda, hızlı bir şekilde her bir oyuğa 500 ul aspire ve uygun bir şekilde, bir 24-çukurlu plaka gözenekleri içinde etiketli ekleyin. Not: süspansiyon hücreleri için, ilk olarak 4 ° C&#39;de santrifüj ile Hücreleri çıkarmak </li><li> 96 oyuklu bir plakaya süpernatan 300 ul hacimde ilave edin ve daha sonra 593 nm&#39;de okundu. </li></ol> Hücre dışı Askorbat 4. Belirlenmesi <ol><li> 593 nm&#39;de kuyuların absorbans değerleri okuyun. </li><li> Hücre içi askorbat tayini için tarif edildiği gibi mg protein başına (ya da milyon hücre başına) nmol askorbat gibi hücre dışı askorbat miktarını hesaplayınAdım 1.5.4 &#39;te. Not: askorbat serbest hücre içi askorbat ile sınırlı değildir, böylece son kullanıcının koşullarını optimize etmek gerekir. </li></ol> LDH Release 5. Belirlenmesi <ol><li> Her bir numunenin hücre dışı çözeltinin geri kalan 200 ul hücre liziz boyutunu belirlemek için bir LDH salma tahlili 32,41 yaparlar. Not: toplam salınabilir LDH% olarak hesaplamak. % 0.1 saponin ile muamele edilmiştir örneklerinden ayrılabilir LDH belirler. </li></ol>

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R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glutamate+aspartate+transporter+(GLAST)+mediates+L-glutamate-stimulated+ascorbate-release+via+swelling-activated+anion+channels+in+cultured+neonatal+rodent+astrocytes.">The glutamate aspartate transporter (GLAST) mediates L-glutamate-stimulated ascorbate-release via swelling-activated anion channels in cultured neonatal rodent astrocytes.</a> Cell. Biochem. Biophys. 65, 107-119 (2012).</li><li>Lane, D. J. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbate+and+plasma+membrane+electron+transport+-+enzymes+vs+efflux.">Ascorbate and plasma membrane electron transport - enzymes vs efflux.</a> Free Radic. Biol. Med. 47, 485-495 (2009).</li><li>Davies, A. R. L., Belsey, M. J., Kozlowski, R. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume-sensitive+organic+osmolyte/anion+channels+in+cancer:+novel+approaches+to+studying+channel+modulation+employing+proteomics+technologies.">Volume-sensitive organic osmolyte/anion channels in cancer: novel approaches to studying channel modulation employing proteomics technologies.</a> Ann. N.Y. Acad. Sci. 1028, 38-55 (2004).</li><li>Novakova, L., Solich, P., Solichova, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HPLC+methods+for+simultaneous+determination+of+ascorbic+and+dehydroascorbic+acids.">HPLC methods for simultaneous determination of ascorbic and dehydroascorbic acids.</a> Trends Anal. Chem. 27, 942-958 (2008).</li><li>Vislisel, J. M., Schafer, F. Q., Buettner, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+sensitive+assay+for+ascorbate+using+a+plate+reader.">A simple and sensitive assay for ascorbate using a plate reader.</a> Anal. Biochem. 365, 31-39 (2007).</li><li>Lane, D. J. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+colorimetric+microplate+ferrocyanide+assay+applied+to+ascorbate-stimulated+transplasma+membrane+ferricyanide+reduction+and+mitochondrial+succinate+oxidation.">A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation.</a> Anal. Biochem. 373, 287-295 (2008).</li><li>Lane, D. J. R., Robinson, S. R., Czerwinska, H., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Na+/H++exchangers+and+intracellular+pH+in+regulating+vitamin+C-driven+electron+transport+across+the+plasma+membrane.">A role for Na+/H+ exchangers and intracellular pH in regulating vitamin C-driven electron transport across the plasma membrane.</a> Biochem. J. 428, 191-200 (2010).</li><li>Corti, A., Casini, A. F., Pompella, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+pathways+for+transport+and+efflux+of+ascorbate+and+dehydroascorbate.">Cellular pathways for transport and efflux of ascorbate and dehydroascorbate.</a> Arch. Biochem. Biophys. 500, 107-115 (2010).</li><li>Laroff, G. P., Fessenden, R. W., Schuler, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electron+spin+resonance+spectra+of+radical+intermediates+in+the+oxidation+of+ascorbic+acid+and+related+substances.">The electron spin resonance spectra of radical intermediates in the oxidation of ascorbic acid and related substances.</a> J. Am. Chem. Soc. 94, 9062-9073 (1972).</li><li>Dringen, R., Kussmaul, L., Hamprecht, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detoxification+of+exogenous+hydrogen+peroxide+and+organic+hydroperoxides+by+cultured+astroglial+cells+assessed+by+microtiter+plate+assay.">Detoxification of exogenous hydrogen peroxide and organic hydroperoxides by cultured astroglial cells assessed by microtiter plate assay.</a> Brain Res. Brain Res. Protoc. 2, 223-228 (1998).</li><li>Lane, D. J. R., Lawen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplasma+membrane+electron+transport+comes+in+two+flavors.">Transplasma membrane electron transport comes in two flavors.</a> Biofactors. 34, 191-200 (2009).</li><li>Lin, S., Lin, D. C., Flanagan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+the+effects+of+cytochalasin+B+on+transport+and+motile+processes.">Specificity of the effects of cytochalasin B on transport and motile processes.</a> Proc. Natl. Acad. Sci. USA. 75, 329-333 (1978).</li><li>May, J. M., Qu, Z. C., Juliao, S., Cobb, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbic+acid+decreases+oxidant+stress+in+endothelial+cells+caused+by+the+nitroxide+tempol.">Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol.</a> Free Radic. Res. 39, 195-202 (2005).</li><li>Avron, M., Shavit, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+simple+method+for+determination+of+ferrocyanide.">A sensitive and simple method for determination of ferrocyanide.</a> Anal. Biochem. 6, 549-554 (1963).</li></ol>

Yazarlar ifşa hiçbir şey yok.

Bu yazıda, kültürlenmiş hücrelerde hücre içi ve hücre dışı bölmelerden türetilen askorbat tayini için iki hızlı, spesifik ve nispeten hassas mikro kolorimetrik tahlilleri mevcut. Tahliller, standart laboratuar ekipmanları ve reaktifler erişim tamamlanabilir. Deney için gerekli olan sadece orta derecede pahalı reaktif L-askorbat doğru analit özgüllük yüksek derecede kazandırmaktadır olarak önemlidir AO vardır. Tahliller, ya da süspansiyon hücreleri (örne?...

1928 1934 1 yayımlanan gazetelerde askorbik asit (C vitamini), ve uzun aranan Albert Szent-Györgyi tarafından &quot;anti-iskorbüt hastalığı faktörü&quot;, ve diğerleri olarak kimlik kimyasal doğanın keşfi tarihinin önemli olaylar vardı biyokimyası. Nitekim, bu keşifler, Szent-Györgyi 1937 yılında Fizyoloji veya Tıp Nobel Ödülü kazandıktan. Hayvan ve bitki fizyolojisi askorbat rolleri giderek genişleyen paketi, yanı sıra insan sağlığını katkıda aktif bilimsel konular olmaya devam soruşturma ve tartışma. L-askorbat bol miktarda fizyolojik bir indirgeme ürünü olan ve memeli sistemlerde enzim kofaktörü ve kollajen hidroksilasyon, karnitin ve norepinefrin biyosentezinin, tirosin metabolizma ve peptid hormonu amidasyonu 2 içeren çok iyi tanımlanmış bir enzimatik reaksiyonlar katkıda bulunur. Intriguingly, montaj evidence askorbat gibi hidroksilasyon ile ilgili prolil ve asparaginil hidroksilazları gibi diğer demir bağımlı dioxygenases, uyarıcı ve hipoksiyayla tetiklenebilen faktörleri (HIFs) 1α ve 3 2α hedeflenmesine bir rol oynadığını göstermektedir. Yeni bir rapor, askorbat, nükleer hidroksilazları, Jumonji C (JmjC) etki proteinlerinin teşvik etmedeki aktivitesi ile kromatin demetilasyonunu etki yoluyla T-hücresi olgunlaşmasında bir rol oynadığını göstermektedir; son olarak adı geçen tam aktivitesi 4 askorbat için gerekli gibi görünmektedir. Gerçekten de, askorbat ve bu tür enzimlerin uyarılması HIF ve kollajen hidroksilazları ve askorbat ile uyarılmaya benzer bir mekanizma ile meydana geldiği görülmüştür. Diğer klasik etkileri arasında, askorbat, suda çözünür zincir açan kök sürükleyici 5 gibi hücresel antioksidasyon ve plazma membranının geri dönüşüm önemli ölçüde katkıda bulunmaktadır α-tokoferol w, 6 α-tokoferoksil radikalin indirgenmesi ile (vitamin E)hich membran lipid peroksidasyonu 7 karşı korunması önemlidir. En memeliler D-glikozdan, askorbat de novo hepatik sentezinin sahip olmasına rağmen Önemli bir şekilde, yüksek primatlar, kobay ve bazı bitler vitamin 8 besin kaynağa bağlıdır. Bu gulo geninin inaktivasyonu nedeniyle, etkilenmemiş memelilerde en orthologues 9-13 oksidaz enzim,-γ-lakton gulono kodlar. Bu enzim, glukoz 13 ila askorbat biyosentezindeki son reaksiyon için gereklidir. Insanlarda bağırsak lümeninden taşıyıcı aracılı emme sonra, askorbat olarak dolaşım sistemiyle vücutta dağıtılır. Vitamin tipik haliyle (konsantrasyonlar tipik olarak geçerli olan plazma konsantrasyonu ile benzer olan eritrositlerin istisna ile) hücre milimolar konsantrasyonlarda mikromolar ve konsantre onun indirgenmiş formu bulunursaEn çok hücre dışı akışkanlar içinde 14,15 entrations (örneğin, 50-200 uM). Fizyolojik koşullar altında, genellikle askorbat askorbil serbest radikali tersine çevrilebilir bir elektron oksitlenme geçirmektedir (AFR, aynı zamanda ya da monodehydroascorbate semidehydroascorbate olarak da bilinir). AFR geri askorbat için hızla bir elektron enzimatik azalma yokluğunda, nispeten kararlı bir radikal 16 iken, iki AFRS bir askorbat ve bir dehydroascorbate (DHA) 9,13,17 için dismutate daha fazla olabilir. Hücresinin iç içinde, askorbat, DHA iki elektron oksidasyon ürünü, hızlı bir şekilde ve glutation-NAD (P) H-bağımlı enzimatik ve enzimatik olmayan reaksiyonları 13 geri askorbat indirgenebilir. Bu klasik demir metabolizması askorbat tek önemli rolü olmayan heme demir 18 diyet emilimini teşvik etmek olduğunu kabul ederken, biz ve diğerleri kanıt s sağladıtrongly bu askorbat düşündüren bu metal metabolizmasında büyük ölçüde genişletilmiş bir rol oynar. İlk olarak, askorbat-dolu hücreler tarafından salınır askorbat hücreleri 19,20 ile transferrine bağlanan demir alımını modüle edilmesinde önemli bir rol oynamaktadır ve çok son kanıtlar askorbat da transferrin bağlı demir alımını modüle olduğunu gösterir gibi görünmektedir hücreleri 21 ile, ve ikinci bir ana fizyolojik demir alım yol 22 karşılık gelir. Askorbat memelilerde 23,24 normal merkezi sinir sistemi fonksiyonu için gereklidir. Birlikte adrenal korteks, hipofiz bezi, timus, retina ve korpus luteum ile, beyin diğer vücut dokularında 23,25-27 askorbat göreli yüksek konsantrasyonlarda bulunur. Buna ek olarak, 28,29 ve nöron astrosit-benzeri hücreler glutamata 30 hem de maruz ascorbat hücre dışı alanı, içine askorbat salınmasını tetiklemek için bilinire glutamat kaynaklı nöronal disfonksiyon 31 karşısında nöronların korunması için düşünülmektedir. Astrositlerden glutamat kaynaklı askorbat serbest mekanizması tam olarak bilinmemekle birlikte, son zamanlarda astrocyte glutamat ve aspartat taşıyıcı (Glast tarafından glutamat alımı nedeniyle şişlik hücrenin katılımını gösteren kanıt sağladı, aynı zamanda bilinen uyarıcı amino asit taşıyıcı izoformdur 1 [EAAT1 ] insanlarda) ve bu askorbat 32 gibi küçük organik anyonları geçiren hacim duyarlıdır osmolite ve anyon kanalları (VSOACs) bunun sonucunda aktivasyonu. VSOAC oluşumunda rol plazma membran borularının 33,34 moleküler kimlikleri tespit edilmesi gerekmektedir. Birçok deneyler, spektrofotometrik fluorometrik tahliller ve kromatografik 35,36 dahil biyolojik numunelerde askorbat tayini için geliştirilmiş olmasına rağmen, seçicilik, hassasiyet, interferenc çok değişkenlik mevcutturkimyasal madde, etkili lineer aralığı ve son nokta analitin stabilite, e. Ayrıca, tahlil seçimini de etkileyecektir diğer önemli faktörleri hız, kullanım kolaylığı ve bu gibi bir yüksek performanslı sıvı kromatografisi (HPLC) aygıtı nispeten özel ekipmanlara erişimi. Burada kültürlenmiş hücrelerde hücre içi askorbat belirlenmesi yanı sıra, kültürlenmiş hücrelerden askorbat-efflux tayini için ayrı bir tahlili için basit ve son derece spesifik mikro kolorimetrik deneyi mevcut. İkinci tahlil nedeniyle sodyum-bağımlı askorbat nakliyecilere (SVCTs) tarafından yayımlanan askorbat hızla yeniden alım hücrelerden askorbat serbest ardı sorunu aşmak için amaçlamaktadır. Bu yöntemlerin her ikisi de önceki yayınlarda 19,20,32,37,38 bazı ortaya çıkmış olsa da, bu el yazması talimatları ve etkin yürütülmesi için kurallar açık bir kümesi sağlar.

<table border="0" cellpadding="0" cellspacing="0" width="678">
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			<td height="60" style="height:60px;width:284px;">Nunc 96-well flat-bottom plates</td>
			<td style="width:135px;">Thermo</td>
			<td style="width:127px;">269620</td>
			<td style="width:132px;">Any flat-bottom 96-well plate can be used</td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:284px;">Refrigerated benchtop microcentrifuge</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;">5415D</td>
			<td style="width:132px;">A non-refrigerated microcentrifuge that has been equilibrated to temperature in a cold room can also be used</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:74px;width:284px;">Refrigerated bench-top centrifuge</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;">5810R</td>
			<td style="width:132px;">Swing-bucket</td>
		</tr>
		<tr height="260">
			<td height="260" style="height:260px;width:284px;">Bio-Rad Benchmark Plus Microplate Spectrophotometer</td>
			<td style="width:135px;">Bio-Rad</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">Any microplate spectrophotometer capable of reading at 593 nm can be used and is recommended. If a filter-based plate reader is used, choose the closest wavelength possible and use the standard-curve method.</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:284px;">Ependorf MixMate (microplate orbital mixer)</td>
			<td style="width:135px;">Eppendorf&#160;</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">This is a very versatile and reliable microplate mixer and works very well for these assays</td>
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			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;width:284px;">General-purpose buffers</td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;width:284px;">Phosphate-buffered saline (PBS), pH 7.4</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:284px;">MOPS-buffered saline (MBS); 137 mM NaCl, 2.7 mM KCl, 15 mM MOPS-Na+, pH 7.3</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">MBS + 5 mM D-glucose (MBS/D)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:284px;">HEPES-buffered saline + 5 mM D-glucose (HBS/D); 137 mM NaCl, 5.2 mM KCl, 1.8 mM CaCl2&#8226;2 H2O, 0.8 mM MgSO4&#8226;7 H2O, 5 mM D-glucose, 20 mM HEPES-Na+, pH 7.3)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Cell permeabilisation buffer (CPB; 0.1% saponin in PBS)</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
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			<td height="20" style="height:20px;">General chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">L-ascorbic acid or sodium L-ascorbate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;">Highest purity preparations should be obtained</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:284px;">Dehydro-L-ascorbic acid (DHA) dimer</td>
			<td>Sigma-Aldrich</td>
			<td>30790</td>
			<td style="width:132px;">Aqueous solutions theoretically yield 2 moles of DHA monomer per mole of DHA dimer</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:284px;">Cytochalasin B</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">C6762</td>
			<td style="width:132px;">Stock solutions prepared in DMSO or ethanol</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:284px;">Ascorbate oxidase (AO)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">A0157</td>
			<td style="width:132px;">Stock solutions (120 U/ml) can be prepared in PBS or MBS and then frozen in aliquots</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Potassium ferricyanide (FIC)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">455989</td>
			<td style="width:132px;">Trihydrate</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Ferene-S (3-(2-Pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5&#8242;,5&#8242;&#8242;-disulfonic acid disodium salt)</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">92940</td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Sodium L-glutamate</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">L-glutamine</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Saponin</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:127px;">47036</td>
			<td style="width:132px;">Prepare a 0.1% stock solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;"></td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">Stock solutions for intracellular ascorbate determination assay</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">3 M sodium acetate (pH 6.0)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">Glacial acetic acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">0.2 M citric acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:284px;">3.3 mM FeCl3 in 0.1 M acetic acid</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">30 mM ferene-S</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:284px;">50% (v/v) acetic acid + 30% (w/v) trichloroacetic acid (TCA)</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stock solutions for ascorbate-efflux assay</td>
			<td style="width:135px;"></td>
			<td style="width:127px;"></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">AO (120 U/ml)</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">2.4 mM ferene-S</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:284px;">0.12 mM FeCl3 in 0.6 mM sodium-citrate</td>
			<td></td>
			<td></td>
			<td style="width:132px;"></td>
		</tr>
	</tbody>
</table>

a rapid and specific microplate assay for the determination of intra- and extracellular ascorbate in cultured cells

C Vitamini (askorbat) sadece son yıllarda ışık gelmiş birçoğu hücre metabolizmasında çok sayıda önemli rol oynar. Beyin askorbat homeostasis için çok önemli gibi görünen bir ilişki - Örneğin, beynin içinde, askorbat nöronlar ve komşu astrositlerden arasında askorbat bisiklet içeren bir nöroprotektif ve nöromodülatör şekilde davranır. Ayrıca, ortaya çıkan deliller güçlü askorbat klasik tanınır daha hücresel ve sistemik demir metabolizmasını düzenleyen bir ölçüde genişletilmiş bir role sahip olduğunu göstermektedir. Normal ve kuralsız hücresel ve organizma fizyolojisi askorbat ayrılmaz rolünün artan tanınması son derece pahalı uzman ekipman için gerek olmadan çalıştırılabilir orta verimlilik ve yüksek hassasiyetli analitik teknikleri bir dizi gerektirir. Burada, orta performans için açık talimatlar, bo tayini için özel ve nispeten pahalı olmayan mikro deneyi sağlarHücre kültüründe th içi ve hücre dışı askorbat.

Kültür Süspansiyon hücreleri içinde hücre içi Askorbat belirlenmesi İlk deneyde (Şekil 1) &#39;de, hücre içi askorbat, önceden yayınlanmış bir prosedüre göre 37 Ferrosiyanat son derece hassas saptaması kullanılarak, ferrosiyanür için ferrisiyanür (örneğin AO duyarlı) indirgeme askorbat-özgü ardından, tespit edilir. Askorbat tespiti ferrosiyanür ile demir ferrik demire indirgenmesi, ardından ferrosiyanür için ferrisiyanür asko...

Watch this Scientific Journal Video about A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells at JoVE.com

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Askorbat sadece son yıllarda ışık gelmiş, birçoğu hücre metabolizmasında çok önemli rol oynar. Burada, orta hacmi, hücre kültürü içinde hem de hücre içi ve hücre dışı askorbat belirlenmesi için özel ve pahalı olmayan bir mikro-tahlili tarif eder.

Kültür Hücrelerinin Intra-ve Ekstrasellüler Askorbat Belirlenmesi için hızlı ve spesifik bir Mikroplate Deneyi

Vitamin C (ascorbate) plays numerous important roles in cellular metabolism, many of which have only come to light in recent years. For instance, within ...

a-rapid-specific-microplate-assay-for-determination-intra

Research

JoVE Journal

Biology

13.4K Görüntüleme.  University of Sydney.  Askorbat sadece son yıllarda ışık gelmiş, birçoğu hücre metabolizmasında çok önemli rol oynar. Burada, orta hacmi, hücre kültürü içinde hem de hücre içi ve hücre dışı askorbat belirlenmesi için özel ve pahalı olmayan bir mikro-tahlili tarif eder. 

Video: A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Doku ve Hücre Sayım ve Canlılık Belirlenmesi

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Biyoloji

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic Testi: yapışkan Hücreler

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

İzolasyon ve Kültür Hücrelerinin ve Fare Doku gelen Mitokondri Fonksiyonel Analizi

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Floresan etiketli Nanopartiküller kullanma Fare, İnsan ve domuz Menşe Canlı hücreler hücre içi Gen İfade Algılama

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

Nükleik Asitlerin süzülmesi İzolasyonu: basit ve hızlı DNA izolasyonu,

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

Hücre kaynaklı Ekstrasellüler Matrix A Hızlı, İzolasyon, Fonksiyonel Çalışmaları Ölçeklenebilir Yöntem ve Analiz

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Transient Expression and Cellular Localization of Recombinant Proteins in Cultured Insect Cells

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and the determination of the biochemical function of proteins at the sub-organismal level. Although baculovirus expression systems are increasingly used for protein production in numerous biotechnological, pharmaceutical, and industrial applications, nonlytic systems that do not involve viral infection have clear benefits but are often overlooked and underutilized. Here, we describe a method for generating nonlytic expression vectors and transient recombinant protein expression. This protocol allows for the efficient cellular localization of recombinant proteins and can be used to rapidly discern protein trafficking within the cell. We show the expression of four&#160;recombinant proteins in a commercially available insect cell line, including two aquaporin proteins from the insect Bemisia tabaci, as well as subcellular marker proteins specific for the cell plasma membrane and for intracellular lysosomes. All recombinant proteins were produced as chimeras with fluorescent protein markers at their carboxyl termini, which allows for the direct detection of the recombinant proteins. The double transfection of cells with plasmids harboring constructs for the genes of interest and a known subcellular marker allows for live cell imaging and improved validation of cellular protein localization.

Nonlytic insect cell expression systems are underutilized for production, cellular trafficking/localization, and recombinant protein functional analysis. Here, we describe methods to generate expression vectors and subsequent transient protein expression in commercially available lepidopteran cell lines. The co-localization of Bemisia tabaci aquaporins with subcellular fluorescent marker proteins is also presented.

Kültürlü Böcek Hücrelerinde Geçici İfade ve Rekombinant Proteinlerin Hücresel Yerelleştirme

Heterologous protein expression systems are used for the production of recombinant proteins, the interpretation of cellular trafficking/localization, and ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

Co-immunoprecipitation tahlil hipoksik koşullarda kültürlü hücrelerden endojen nükleer proteinler kullanarak

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...