yazarlar normal beyin numuneleri sağlamak için İnsan Beyin ve Omurilik Sıvısı Kaynak Merkezi (Los Angeles, CA) müteşekkiriz. Bu çalışma McGregor Vakfı&#39;nın desteği ve Cumhurbaşkanı İhtiyari Fonu (Case Western Reserve Üniversitesi) ile Ulusal Sağlık Enstitüleri (NIH) R01NS062787, CJD Vakfı, İttifak BioSecure yanı sıra Üniversite Yaşlanma Merkezi ve Sağlık tarafından desteklenen .

1. Beyin Homojenat ve Deterjan-Çözünür (S2) ve çözünemeyen (P2), fraksiyonlar hazırlanması <ol><li> 100 mg insan dondurulmuş beyin dokusu almak ve (10 mM Tris, 150 mM NaCl,% 0.5 Nonidet P-40 (NP-40),% 0.5 EDTA, pH 7.4) 100 ul lizis tamponu ilave edin. </li><li> Bir akülü motor tarafından tahrik havaneli kullanarak buz üzerinde homojenize, bu da 20 olan 5 dk için kuru buz içinde dondurmak ve motor tarafından ilk ve sonra eller tarafından tahrik havaneli kullanarak tekrar homojenize ve 300 ul veya 800 ul lizis tamponu (ekleyin % veya 10% toplam beyin homojenat, sırasıyla). </li><li> 4. 10 dakika ° C benchtop santrifüj kullanarak süpernatant (S1) toplamak için 1000 xg&#39;de% 20 veya% 10 toplam beyin homojenat santrifüjleyin. </li><li> 4 ° C&#39;de 1 saat boyunca bir SW55 rotor içinde 35,000 rpm (100,000 xg) (Beckman Coulter, Fullerton, CA)% 10 S1 Ultrasantrifüj Temiz bir tüp içine deterjan çözünebilen fraksiyonu içeren süpernatan (S2) aktarın. Çözünmeyen deterjan içeren granül süspanseya da 5-kat konsantre hazırlama - 100 ul ya da 10 yapmak için 200 ul lizis tamponu içinde fraksiyonu (P2). </li><li> PK-sindirim, S2 ve P2 fraksiyonların her ikisi için de, 1 saat boyunca 37 ° C &#39;de 50 ug / ml&#39; de PK aynı miktarda örnek inkübe edilir. 5 mM ve PK sindirim sonlandırmak için SDS numune tamponu (% 3 SDS, 2 mM EDTA,% 4 β-merkaptoetanol,% 10 gliserol, 50 mM Tris, pH 6,8) ve eşit hacimde son konsantrasyon phenylmethylsulfonyl fluorür ekleyin. 10 dakika için kaynatılır ve numuneler 5 dakika için oda sıcaklığında soğumasına onları. Onlar Western blot için hazırız. </li></ol> 2. Sukroz Adım Degradeler Hız ve Sedimantasyon <ol><li> Buz üzerinde 30 dakika için H2O içinde% 2 sarkosyl eşit bir hacim ile 450 ul% 20 S1 inkübe edin. </li><li> 0.5 ml% 60 her biri,% 30,% 25,% 20,% 15, ve en fazla 5 ml kapasiteli bir Beckman tüp içine% 10 sakaroz ekleyin. </li><li> 10-60% sakaroz adım degradelerin üstüne S1 0.4 ml yükleyin. </li><li> 4 santrifüjleyin4, 1 saat boyunca 8000 rpm (200,000 xg, SW55 rotor), ° C. </li><li> Her tüpün üst 283 ul fraksiyonu toplayın ve toplam 12 fraksiyon elde. </li><li> Yeni bir Eppendorf tüpüne her bir fraksiyon 20 ul al, 10 dakika boyunca 20 ul örnek bir tampon, ve kaynatılır. </li><li> Kaputu 4 dakika Serin örnekleri, 1 dk ve vorteks onlar için 1.000 xg santrifüj. Bir ön döküm jel üzerine örnekleri yerleştirin. </li></ol> 3. Boyut Dışlama Kromatografisi <ol><li> PrP moleküllerin oligomerik durumunu belirlemek için bir 1 x 30 cm sütun Superdex 200 HR boncukları (Pharmacia, Uppsala, İsveç) kullanın. </li><li> Albümin Dekstran mavi (2.000 kDa), tiroglobulin (669 kDa), apoferritin (443 kDa), β-amilaz (200 kDa), alkol dehidrogenaz (150 kDa), dahil olmak üzere yedi moleküler kütle belirteçleri (Sigma, St Louis, MI) ( 66 kDa), karbonik anhidraz (29 kDa) 200 ul numune hacimlerindeki Sigma tarafından tavsiye edilen konsantrasyonlarda bağımsız olarak yüklenir. </li><li> Mavi Dext elusyon hacminderan ürün komutu tarafından sağlanan boşluk hacmi (V 0 = 8.45 ml) ve toplam hacmi (V t = 24 ml) belirlemek için kullanılır. Pik elüsyon hacmi (V e) ve kromatogram fraksiyonel kestiği hesaplanır. - / (V t - V 0) K av = (V 0 V e): K av, dağılım katsayısı (örnek davranışını tanımlayan), denklem kullanılarak hesaplanır. Kalibrasyon eğrisi standartları 8 günlük MW karşı protein standartlarının K av çizerek belirlenir. </li><li> Farklı FPLC fraksiyonları ele çeşitli PrP türlerin molekül ağırlığı (MW) çeşitli standartları jel filtrasyon ile oluşturulan bir kalibrasyon eğrisine göre değerlendirilir. </li><li> Her çalışma için sütuna 1% sarkosyl içeren lizis tamponu 200 ul S1 enjekte. </li><li> Kromatografi 0.25 ml / dk ve fraksiyonunun bir akış hızında, bir FPLC sistemi (GE Healthcare) içinde gerçekleştirilirin 0.25 ml &#39;lik her bir fraksiyon toplayıcısı (Amersham Biosciences, RediFrac) kullanılarak toplanmıştır. </li><li> 2 saat boyunca -20 ° C&#39;de 0.5 ml önceden soğutulmuş metanol ile 0.125 mL, her fraksiyonda inkübe edin. </li><li> 4. 30 dakika ° C benchtop mikrosantrifüj en çok 13.000 xg&#39;de santrifüjleyin. Süpernatant atılır, oda sıcaklığına kadar soğutulur 10 dakika için kaynar, yukarıda da belirtildiği gibi 20 ul SDS numune tamponu içinde tekrar süspansiyon pelet, bir ön-dökme jel üzerine yerleştirin. </li></ol> 4. G5p tarafından PrP Capture <ol><li> G5p molekülü (100 mikrogram) (nazik Portsmouth, UK Üniversitesi&#39;nden Dr. Geoff Kneale ve John McGeehan tarafından sağlanan) 7 konjuge x 10 8 tosil 1 ml 100 mikrogram G5p ve 350 ul G5p boncuk inkübe ederek manyetik boncuk aktive 20 saat süre ile 37 ° C &#39;de bir fosfat-tamponlu salin (PBS). Dış manyetik kuvvet tarafından Eppendorf tüpleri yan duvarına G5p-boncuk çekin ve sonra tüm çözüm çıkarın. 1 ml PBS içeren boncuklar ile yıkayın% 0.1 sığır üç defa için albümin (BSA) serum. </li><li> Spesifik olmayan bağlanma engellemek için 5 saat için 0.2 M Tris-HCI, 37% 0.1 BSA ihtiva eden pH 7.4 &#39;° C&#39;de 1 ml G5p boncuk ile konjuge inkübe ve sonra PBS içinde 1 ml% 0.1 BSA ihtiva eden boncuk ile yıkayın üç kez yukarıda da belirtildiği gibi. Çözüm çıkarın ve boncuklar içine PBS 1 ml ekleyin. Hazırlanan G5p boncuklar 4 ° C &#39;de en azından 3 ay boyunca stabil </li><li> G5p ile PrP yakalama bağlayıcı tampon, 1 ml 60 ul G5p konjuge boncuklar (10 ug protein / 6 x 10 7 boncuk) (% 3 Tween 20,% 3 NP-S1 ile 100 ul fraksiyon ya da P2 inkübe edilmesiyle gerçekleştirilir PBS, pH 7.5 &#39;te 40). </li><li> Oda sıcaklığında 3 saat boyunca sabit dönüş ile inkübasyondan sonra, PrP-içeren G5p boncuk çözeltisi içinde çözülmüş bütün moleküller kolayca çıkarılmasına izin veren, harici bir manyetik kuvvet ile Eppendorf tüpleri yan duvarına karşı çekilir. </li><li> Yıkama tamponu ile üç yıkama (% 2 Tween-20 ve PBS içinde% 2 NP-40, pH 7 takiben.5), G5p boncuklar 95 toplanır ve ısıtılır SDS numune tamponu içinde 5 dakika (% 3 SDS, 2 mM EDTA,% 10 gliserol, 50 mM Tris-HCl, pH 6,8) için ° C. </li><li> Oda sıcaklığında 5 dakika için örnekler soğutulduktan sonra, numuneler, oda sıcaklığında 5 dakika boyunca 1000 x g&#39;de santrifüj edilir. Süpernatantlar Western blot için hazırız. </li></ol> 5. Western Blotting <ol><li> Yük örnekleri ~ 80 dakika için 150 V az% 15 mM Tris-HCl, Ölçütleri önceden dökme için jeller (Bio-Rad) üzerine SDS numune tamponu içinde kaynatılır. </li><li> Jelleri üzerinde proteinleri 70V az 2 saat süreyle PVDF transfer edilir. </li><li> % 5 süt içinde bloke sonra zarlar Tris-tamponlu salin% 0.01 Tween-20 ihtiva eden (TBS-T), membranlar 1E4 (1, birincil monoklonal ya da poliklonal antikor 3F4 (1/40.000) ile oda sıcaklığında 2 saat süreyle inkübe edilmektedir : 1000), anti-N 8 (1:6,000), ya da anti-C PrP molekül tarama için 8 (1:6,000). </li><li> HRP-konjüge at koyun anti-fare IgG ile inkübasyon sonrasında1:3000, ya da eşek anti-tavşan IgG 1:3,000 da PrP bantları, imalatçının protokolüne göre, ECL Plus kullanılarak Kodak film üzerinde görüntülenmiştir. </li></ol> 6. Temsilcisi Sonuçlar PrP C çoğu S2 fraksiyonu (Şekil 1) elde edildi rağmen Sporadik CJD&#39;li numuneleri ile karşılaştırıldığında, iPrP C sinin küçük bir miktarı normal beyin içinde P2 fraksiyonunda saptanmıştır. Tam uzunlukta ve N-terminal olarak kısaltılır türler de dahil olmak üzere toplam PrP yaklaşık olarak% 5-25 için önceden 8, iPrP hesaplar belirtildiği gibi. Sukroz adım gradyanı sedimentasyon kullanarak Analizleri olmayan CJD beyninden PrP C çoğunu 1-3 üst kesirler ele iken, PrP küçük miktarlarda da normalde büyük agrega 8 (Şekil içeren alt fraksiyonları 9-11 saptandığını ortaya koydu 2). PrP Sc türlerin çeşitli çaldımonomerlerden ing, daha büyük agregalar küçük oligomerler, Creutzfeldt-Jakob hastalığı (Şekil 3A) ile beyindeki jel filtrasyon ile izole edildi. Bununla birlikte, 2,000 kDa molekül ağırlığı daha yüksek olan büyük agrega küçük bir miktar, aynı zamanda, normal beyin çözünmeyen fraksiyon (Şekil 3C) olarak tespit edildi. Ayrıca, dimerleri ve PrP C tetramers yalnızca çözünmeyen fraksiyonlar hem de çözünebilir fraksiyon (Şekil 3B ve 3C) tespit edilememiştir. PK ve PNGase tedaviden sonra, G5p tarafından yakalanan PrP PrP97-105 8 karşı 1E4 antikoru ile tespit edildi. Üç PK dayanıklı çekirdek parçası olarak adlandırdığı PrP * 20, PrP * 19 ve PrP * 7 sırasıyla ~ 20 kDa, ~ 19 kDa ve ~ 7 kDa (Şekil 4, sol panel) de göç, tespit edildi. 1E4 antikor bir sekans tanım olan bir sentetik peptid ile önceden inkübe edildi, ancak, herhangi bir PrP tespit1E4 ile tespit bantlar PrP parçası bulunmaktadır gösteren 1E4 epitopu (Şekil 4, orta panel) ile ical. Ayrıca, anti-C antikor ~ 18 kDa az iki farklı geçiş PrP fragmanları tespit (PrP * 18) ve PrP ek olarak ~ 12-13 kDa (PrP-CTF12/13), * 20 (Şekil 4, sağ panel). <img alt="Şekil 1" src="/files/ftp_upload/3788/3788fig1.jpg" /> Şekil 1. IPrP C ve iPrP Sc Algılama. 1 saat boyunca 37 ° C&#39;de toplam reaksiyon hacmi 1/10 PNGase F ile tedaviden sonra protein, tam uzunluklu ya da N-terminal olarak (S2 ve P2) çözünür ve çözünür olmayan fraksiyonlarının PrP tür kesilmiş izole dan glukanlardır kaldırmak için normal kontrol (CTL) ve sporadik CJD (sCJD) beyin örneklerinde ultrasantrifiij ile PrP23-40 (orta panel) karşı PrP106-112 (sol panel), anti-N karşı 3F4 ile tespit edildi ve PrP karşı anti-C220-231 (sağ panel). Büyük miktarda S2 içinde mevcut iken, CTL örneklerde, PrP küçük bir miktar, P2 olarak tespit edilir. Buna karşılık, daha PrP sCJD örnekleri S2 daha P2 tespit edilir. <img alt="Şekil 2," src="/files/ftp_upload/3788/3788fig2.jpg" /> Şekil 2. Sükroz adım Degradelerde PrP sedimantasyon. 1 olmayan CJD beyin numunesini S1 11 bireysel kesirler PrP Batı 3F4 ile blot tespit edildi. PrP C çoğu 1-3 üst kesirler tespit edilmesine karşın, PrP küçük miktarlarda da dip kesirler 9-11 gözlendi. Ayrıca, üst ve alttan PrP bant deseni farklıdır: alt fraksiyonları ele PrP baskın alt band varken iyi kesirler ele PrP baskın bir üst bant vardır. A PK-tedavi PrP Sc leke sağ tarafında bir kontrol olarak yüklendi. <img alt = &quot;Şekil 3&quot; src = &quot;/ files/ftp_upload/3788/3788fig3.jpg&quot; /&gt; Şekil 3. Çözünür ve çözünmez PrP C oligomerlerinin Algılama. Normal insan beyninden çözünür ve çözünmez PrP C ultra-santrifüj ile ayrılır ve daha sonra, sırasıyla, jel filtrasyon tabi tutuldu. Bireysel fraksiyonların molekül boyutları moleküler ağırlığı bilinen bir grup çalışan ile ölçüldü. SCJD beyin numuneleri (A) PrP Sc türler. PrP türlerin iki nüfus tespit edilmiştir: jel filtrasyon fraksiyonu 49 - kesirler 27-33 büyük agrega içerir oysa 65, monomerler ve küçük oligomerler içerir. Normal kontrol çözünebilir fraksiyon (S2), (B) ve çözünmeyen bölüm (P2), (C) PrP C tür tespit edildi. PrP 3F4 antikoru ile sondajlanan edildi. Dimerleri (fraksiyon 55) ve PrP tetramerleri (fraksiyon 51), P2, aynı zamanda, normal beyin sampl S2 olarak değil, sadece tespit edildiES (B ve C). Büyük agregalar sadece normal örnekleri (C) P2 saptandı. <img alt="Şekil 4," src="/files/ftp_upload/3788/3788fig4.jpg" /> Şekil 4. Normal insan beyninden G5p zenginleştirilmiş hazırlıkları çeşitli PK dirençli iPrP fragmanlarının tespiti. G5p ile zenginleştirilmiş örnekleri Western (sol panel), 1E4 1E4 epitopu (orta panel) için özdeş bir sekans vardı bir sentetik peptid ile 1E4 önceden inkübe ile problama kurutma öncesinde PK ve PNGase F ile muamele edilmiş ve anti-C (edildi sağ panel). 1E4 üç PK dayanıklı PrP fragmanları adlandırdığı tespit PrP * 20, PrP * 19 ve PrP * 7 (sol panel). Peptit ile 1E4 engelleme sonra, hiç PrP res 1E4 ile tespit bantları PrP parçaları olduğunu belirten, (orta panel) saptandı. Anti-C, iki yanı PK dayanıklı PrP fragmanları adlandırdığı ortaya PrP * 18 ve PrP-CTF12 /13, PrP * 20 ek olarak.

<ol>
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Çıkar çatışması ilan etti.

Burada bildirilen yaklaşımların birleşimi normal insan beyninin sürekli çözünmez PrP C agrega ve çözünür PrP C oligomerler ayırır. Bir saat boyunca 100,000 x g&#39;da santrifüjü gibi geniş ölçüde çözünür PrP C den 14 çözünmez PrP Sc ayrılması için kullanılmış olan klasik bir yöntemdir. Bu, uyarılar bir etkili olsa da santrifüj sonrasında (S2) süpernatant çekme sırasında kirlenmesini önlemek içindir. IPrP C jel pr...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Phenylmethylsulfonyl florür </td><td> Sigma-Aldrich </td><td> P7626 </td><td> 2-propanol içinde 72 mM </td></tr><tr><td> Proteinaz K </td><td> Sigma-Aldrich </td><td> P2308 </td><td> H2O içinde 2 mg / ml </td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> MWGF200 </td><td></td></tr><tr><td> LE-80K, SW55 rotor ultrasantrifüjdeki </td><td> Beckman Coulter </td><td> Parça No 365668, 356860 </td><td></td></tr><tr><td> Superdex 200 HR boncuk </td><td> GE Healthcare </td><td> 17-1088-01 </td><td></td></tr><tr><td> AKTA FPLC sistemi </td><td> GE Healthcare </td><td> 18-1900-26 </td><td></td></tr><tr><td> Molekül ağırlığı belirteçler </td><td> Sigma-Aldrich </td><td> MWGF200 </td><td> Çeşitlidir </td></tr><tr><td> Dynabeads M-280 manyetik boncuk </td><td> Invitrogen </td><td> 143-01 </td><td></td></tr><tr><td> 3F4 antikoru </td><td> Covance </td><td> SIG-39600 </td><td></td></tr><tr><td> Antikor 1E4 </td><td> Hücre Bilimleri </td><td> M1840 </td><td></td></tr><tr><td> Hazır jel% 15 mM Tris-HCl, önceden dökme jeller </td><td> Bio-Rad </td><td> 345-0020 </td><td></td></tr></tbody></table>

isolation of soluble and insoluble prp oligomers in the normal human brain

Prion hastalıklarının patogenezinde merkezi bir olay, onun patojenik izoformu PrP Sc 1 içine konak-kodlanmış hücresel prion proteini PrP C bir dönüşüm içerir. PrP-Sc deterjan çözünmez agregaları ve PK 2-6 karşı kısmen dirençlidir ise PrP C, deterjan çözünebilen ve proteinaz K (PK)-sindirim duyarlıdır. PrP Sc PrP C dönüştürme protein β-tabaka yapıları α-sarmal bir konformasyon geçiş dahil olduğu bilinmektedir. Ancak, in vivo yolu içinde çok az anlaşılmıştır. Bir geçici endojen PrP Sc, ara PrP * veya &quot;sessiz prion&quot;, bulaşmamış beyin 7 tespit edilmesi henüz. Biyofiziksel ve biyokimyasal yaklaşımların bir arada kullanarak, bulaşmamış memeli beyin ve nöronal kültürlü gelen çözünmez PrP C agrega (belirlenmiş iPrP C) tespithücreler 8, 9. Burada, deterjan tampon, sukroz adım degrade sedimantasyon, boyut dışlama kromatografisi, özellikle yapısal değişmiş PrP formları, 10 ve PK-tedavi bağlamak gen 5 proteini (G5p) tarafından iPrP zenginleşmesine Ultrasantrifügasyon dahil olmak üzere bu yöntemlerin ayrıntılı prosedürleri açıklar. Bu yaklaşımların birleşimi çözünmeyen PrP Sc ve PrP C büyüklükler değil, aynı zamanda normal insan beyninden çözünür PrP C oligomerler sadece ayırır. Protokolleri enfekte beyinleri ve enfekte olmayan beyninden iPrP C PrP Sc hem izole etmek için kullanılan oylandı Burada açıklanan bu yana, fizikokimyasal özellikleri, nörotoksisite ve iki izoformu arasındaki enfektivite farklılıkları karşılaştırmak için bir fırsat verin. Böyle bir çalışmanın büyük ölçüde bulaşıcı proteinli patojenlerin anlayışımızı geliştirecektir. IPrP C fizyolojisi ve patofizyolojisi halen belirsizdir. Özellikle, yeni-idyazısından İnsan prion hastalığı proteaz duyarlı prionopathy değişken olarak adlandırılan, biz iPrP C 11, 12 ile immunoreaktif davranış ve parçalanma paylaşan yeni PrP Sc bulundu. Ayrıca, biz son zamanlarda iPrP C Alzheimer hastalığı 13 amiloid-β proteini ile etkileşime ana tür olduğunu göstermiştir. Aynı çalışmada, bu yöntemlerin diğer nörodejeneratif bozukluklar ile ilgili olmayan prion proteininin agregalar için uygulama düşündüren, Alzheimer hastalığının 13 Abeta agrega ve oligomerler izole etmek için kullanılmıştır.

Watch this Scientific Journal Video about Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain at JoVE.com

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

Hücresel prion proteini yeni bir tür (PrP<sup&gt; C</sup&gt;) Son zamanlarda burada açıklanan yöntemleri kullanarak bulaşmamış insan beyni saptanmıştır. Bunların bazıları, aynı zamanda, insan beyninden diğer katlanan protein büyüklükler izole yararlıdırlar Bu yöntemler, çeşitli PrP tür izole etmek için de kullanılabilir.

Normal İnsan Beyin Çözünür ve çözünmez PrP Oligomerleri İzolasyonu

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

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Research

JoVE Journal

Medicine

10.7K Görüntüleme.  Case Western Reserve University School of Medicine. Hücresel prion proteini yeni bir tür (PrP C) Son zamanlarda burada açıklanan yöntemleri kullanarak bulaşmamış insan beyni saptanmıştır. Bunların bazıları, aynı zamanda, insan beyninden diğer katlanan protein büyüklükler izole yararlıdırlar Bu yöntemler, çeşitli PrP tür izole etmek için de kullanılabilir.

Video: Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Karboksi Flöresein sukkinimidil ester (KAKE) Kullanarak İnsan Glioblastoma Yavaş-bölme Hücreleri Tanımlama ve İzolasyonu

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Tıp

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson's disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 1. Because ageing is the most important risk factor, cases of PD will increase during the next decades 2. Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD 3-11.
After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.
Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson&#039;s Disease

Fibroblasts from patients carrying mutations in Parkinson's disease-causing genes represent an easily accessible ex vivo model to study disease-associated phenotypes. Live cell imaging gives the opportunity to study morphological and functional parameters in living cells. Here we describe the preparation of human fibroblasts and subsequent monitoring of mitochondrial phenotypes.

Parkinson Hastalığı Alanında Mitokondriyal Fenotiplerinin İzleme İlköğretim İnsan Fibroblast Kullanımı

Parkinson's disease (PD) is the second most  common movement disorder and affects 1% of people over the age of 60 1. Because ageing is  the most important ...

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Lezyon Explorer: Doğru ve Güvenilir MR türetilmiş Alzheimer Hastalığı Volumetrics ve Normal Yaşlı Bir Video-güdümlü, Standart Protokol

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

Floresan SapC-DOPS Nanovesicles kullanma Beyin Tümörleri ve Artrit Vivo Optik Görüntüleme

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Bir olarak Beyin Microslices arasında excitotoxic uyarılması<em&gt; In vitro</em&gt; İnme Model

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

İnsan Epileptik Ameliyat sonrası Kortikal Doku Çok elektrot Dizi Kayıtlar

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

İzolasyon, Kriyoprezervasyon ve Kültür Klinik Uygulamaları İnsan Amniyon Epitel Hücreleri

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Çoklu-parametre floresans ile aktive edilen hücre sınıflandırma İnsan lenfatik endotel hücreleri İzolasyonu

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

Derin Beyin Uyarım ile Saccadometry kullanarak normal ve patolojik beyin fonksiyonlarını incelemek için

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

Mechanical and Controlled PRP Injections in Patients Affected by Androgenetic Alopecia

23 patients (18 male and 5 female) aged 21-70 years who displayed male pattern hair loss (MPHL) in Stage 1 to Stage 5 as determined by the Norwood-Hamilton classification scale, and female pattern hair loss (FPHL) in Stage 1 to Stage 2 as determined by the Ludwig classification scale, were treated with non-activated autologous platelet-rich plasma (A-PRP). Autologous blood (55 mL) was harvested using sodium citrate as an anticoagulant. A-PRP (23 mL) was produced for all cases using a closed system according to the transfusion service protocol. Following centrifugation (260 x g for 10 min) the A-PRP was inserted in a laser light selector device, and after the centrifugation, 9 mL of A-PRP was collected.
The scalp of the patients affected by androgenetic alopecia (AGA) was divided into four areas (frontal, parietal, vertex, and occipital); local anesthesia was not performed. Interfollicular A-PRP injections (0.2 mL x cm2) were performed by controlled and mechanical injections scheduled at a depth of 5 mm using a medical injector gun. Treatment sessions were performed with a 30-day interval. For each patient, three treatment sessions were performed.
PRP was injected in the androgen-related areas of scalp affected by hair loss. Placebo (normal saline solution) was loaded in another syringe (10 mL) and injected on the adjacent side in a similar fashion.

This paper aims to describe the interfollicular autologous platelet-rich plasma injections (0.20 mL x cm2) in the scalp of 23 patients affected by androgenetic alopecia at a depth of 5 mm using a medical injector gun equipped with a 30 gauge needle, in three sessions.