Bu çalışma, Avustralya Ulusal Sağlık ve Tıbbi Araştırma Konseyi, Star Bilimsel Vakfı (Avustralya), Tourette sendromu Derneği (ABD) tarafından desteklenen, Trish Multipl skleroz Araştırma Vakfı ve Multipl Skleroz Araştırma Avustralya, Petre Vakfı (Avustralya), Rebecca L. Cooper Tıbbi Araştırma Vakfı (Avustralya). Hepimiz hasta ve bizim çalışma için örnekleri verilen aile üyelerine teşekkür ederim.

<html> 1.. Strateji pIRES2-EGFP Vektör Kodlama D2R veya NMDAR Construct altklonlamıştır <ol><li> İnsan D2R veya NMDAR alt biriminin 1 (NR1) tam uzunluktaki cDNA klonunu elde edilir. </li><li> Böyle bir durumda birlikte tanımlanması antijenleri ve GFP hem de mümkün kılan bir gelişmiş yeşil floresan protein, bir iç ribozom giriş sahası (IRES) kontrolü altında (GFP) raportör ile transmembran proteinlerin ekspresyonu için uygun olan pIRES2-EGFP gibi bir ifade vektörü, seç ayrı ayrı hücreler. </li><li> PIRES2-EGFP vektörü içinde insan cDNA alt-klonlanmıştır. <ol><li> , CDNA alt klonlamak, uygun kısıtlama enzimleri kullanmak için (insan D2R cDNA örneğin Nhel ve Xhol için). </li><li> Ligatı kesilmiş cDNA kısıtlı pIRES2-EGFP vektörü içinde yerleştirin. </li><li> Dizisi vektör doğru sırasını içerip içermediğini kontrol etmek pIRES2-EGFP D2R veya NMDAR vektörü bağlandı. </li><li> Arındırmak ve transfeksiyon daha sonra kullanmak için pIRES2-EGFP D2R veya NMDAR vektörün yükseltilmesi. </li></ol></li></ol>itle &quot;&gt; 2. Nöronal Antijen İfade için HEK293 HÜCRE NAKLİ <ol><li> Taze Dulbecco Modified Eagle Medium (1x) (DMEM) 4.5 g / L D-glikoz, L-glutamin ve 110 mg,% 10 fetal sığır serumu (FBS) ile desteklenmiş / L sodyum piruvat ile birlikte ile doku kültürü şişelerinde Kültür HEK293 hücreleri, 2 mM GlutaMAX ve 50 ug / ml Gentamisin. </li><li> Tripsin ve transferi bir konik tüp kullanılarak HEK293 hücreleri ayırın. <ol><li> 6 dakika boyunca 250 xg&#39;de hücreler santrifüj ile yıkayın ve taze bitmiş DMEM içinde bir hücre pelletini. </li></ol></li><li> Yaklaşık 8 x 10 5 hücre / göz ve kültür, gece boyunca 2 ml DMEM içinde 37 ° C de ve% 5, bir kuluçka makinesi içinde CO2 de hücreleri ve tohum 6 kuyucuğu sayılır. </li><li> Bu yaklaşık% 70 ortak akışkanlığa eriştiklerinde sonra, pIRES2-EGFP NR1 (HEK293 NMDAR + hücreleri), pIRES2-EGFP D2R (HEK293 D2R + hücreleri) ile HEK293 hücreleri transfekte etmek, ve pIRES2-EGFP (HEK293 CTL hücreleri vektör kontrolü) aşağıdaki gibi.Sonra tazminat için bazı untransfected HEK293 hücreleri tutun. <ol><li> 2.5 ug DNA, 200 ul% 0.9 sodyum klorür ve 4 ug / ml polietilenimin / oyuk (her biri 2 ml taze tam DMEM içeren) içeren bir transfeksiyon karışımı gerekli hacmi hazırlayın. </li><li> Hemen 10 saniye boyunca girdap karışımı ve uygun oyuklara transfeksiyon karışımı hacmi ilave edilmeden önce, 10 dakika süre ile oda sıcaklığında (RT) enkübe edilir. </li><li> , Kapak plakası Parafilm ile yanlarını doğal olarak kaplayan ve hücre transfeksiyonu yardımcı olmak için 5 dakika boyunca 280 x g &#39;de santrifüj. </li><li> Parafilm çıkarın ve sonra 37 ° C de kültüre inkübatöre koyun </li><li> Taze tam DMEM ile 18 saat sonra kültür ortamı değiştirin ve başka bir 72 saat boyunca kültür korumak. </li><li> İsteğe bağlı: 72 saat post-transfeksiyon, transfekte edilmiş hücreler, poliklonal stabil transfektan elde etmek amacıyla, 250 ug / ml Genetisin ile desteklenmiş tam DMEM içinde kültür tarafından seçilebilir. </li></ol></li></ol><pclass = &quot;jove_title&quot;&gt; 3. Nöronal Antijenleri Yüzey antikor tespiti için Cytometry hücre bazlı bir deneyde Akış <ol><li> 37 ° C&#39;de yaklaşık 5 dakika boyunca 500 ul / well Versene ile kuluçkaya yatırılması ile HEK293 NMDAR +, HEK293 D2R + ve HEK293 hücreleri CTL ayırın <ol><li> PBS içinde süspanse 2ml/well (= Ca 2 + / Mg2 +)% 2 FBS (PBS / FBS) ve 15 ml veya 50 ml konik bir tüp transfer ile takviye edilmiştir. </li><li> 6 dakika boyunca 250 xg&#39;de santrifüj ile hücreler yıkanır ve 15-50 ml PBS / FBS hücre pelletini. Yıkama 2x tekrarlayın. </li><li> Hücre sayımı ve daha sonra 1 x 10 6 hücre / ml &#39;de PBS / FBS içerisinde yeniden süspanse edin. </li></ol></li><li> HEK293 D2R + veya HEK293 NMDAR + hücreleri, hem de CTL HEK293 hücreleri, her bir hasta numune ya da birincil antikor ile inkübe edilmesi olacağı dikkate alınarak, akış sitometri elde edilmesi için 96-çukurlu bir şablon tasarımı. </li><li> 50,000 hücre / biz bir V-tabanlı 96 oyuklu bir plaka içerisinde tohum hücrelerill alıcı şablon sitometri akışına göre. </li><li> 5 dakika boyunca 450 x g &#39;de 96-yuvalı plaka santrifüj ve nazikçe bir elektronik çok kanallı pipet ya da elle kullanarak süpernatan, bir açı ile plaka eğme ve hücre pelletini aspire dikkat alarak Pelet hücreleri. </li><li> HEK293 D2R +, HEK293 NMDAR + ve karanlıkta, oda sıcaklığında 1 saat boyunca inkübe edilmiş HEK293 hücreleri CTL: <ol><li> Yüzey antijen ekspresyonunu değerlendirmek için birincil antikorunun seri dilüsyonları. </li><li> Antikoru belirlemek amacıyla hasta örnekleri. </li></ol></li><li> Örneğin, tazminat kontrolleri sitometri uygun akışı kullanın. boyanmamış transfekte edilmemiş HEK293 hücreleri, GFP + HEK293 hücreleri (AF647 -) ve transfekte edilmemiş AF647 + HEK293 hücreleri (GFP). </li><li> 5 dakika boyunca 450 x g santrifüj ile hücreler yıkanır ve 170 ul PBS / FBS hücre pelletini. Yıkama adımı iki kez daha tekrarlayın. </li><li> 1 saat boyunca inkübe hücreleri(anti-NR1 ya da anti-D2R primer antikorlar hasta serum ve anti-fare IgG anti-insan IgG) AF647-konjuge sekonder antikor 1:100 seyreltme ile karanlıkta, oda sıcaklığında r. </li><li> Adım 3.7 &#39;de olduğu gibi, hücreler üç kez yıkayın ve hücre devir akış sitometrisi için 35 ul PBS / FBS içerisinde yeniden süspanse edin. </li><li> (BDLSRII) sitometresi akış yüksek verim sampler (HTS) ayarlayın. </li><li> Edinimi şablonun sitometri akışına göre 10.000 olaylar / kuyu edinin. </li><li> İhracat ve sonra bu FlowJo v7.5 gibi yazılım paketinin sitometri akışını kullanarak analiz için verileri analiz: <ol><li> İlk olarak, ileri (boyut) ve yan (granüllü yapı) göre kapı canlı HEK293 hücreleri dağıtır. </li><li> Yalnızca transfekte hücreleri analiz etmek için yüksek GFP-pozitif göre daha fazla kapı canlı HEK293 hücreleri. </li><li> Canlı GFP-pozitif hücreler içinde AF647 kanalın ortalama floresan yoğunluğu (MFI) belirler. </li><li> Analiz için Excel veya prizma içine İhracat verileri: <ol><li> Plot cMFI karşı birincil antikor (anti-NMDAR veya anti-D2R antikor) oncentrations, bu antikorlar ile inkübe hücrelerden elde. </li><li> Her bir hasta ve kontrol örneği için, sırası ile, HEK 293 D2R + ya da HEK293 NMDAR + hücrelerinin MFI gelen CTL HEK293 hücrelerinin MFI çıkarılarak ΔMFI belirler. </li><li> Kontrol kohort ortalama ΔMFI yukarıdaki üç standart sapma ekleyerek antikor pozitifliğinin eşiğini hesaplayın. </li><li> Arsa bireysel örnekler bir grafik üzerindeki serum türüne göre gruplandırılmış ve bir çizgi halinde eşiği temsil etmektedir. </li></ol></li></ol></li></ol>

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Çıkar çatışması: Bir patent otoantikorlar için hedef olarak D2R iddia FB ve RCD (Sydney Üniversitesi) tarafından dava açılmıştır.

Bu çalışma, bir yüksek verimli bir örneği kullanarak, belirli hücre yüzeyi proteinlerini nöronal hedef antikorları tespit etmek için bir canlı hücre bazlı bir deneyde akış sitometrisi yeni bir uygulamasını tarif etmektedir. Bu tekniği kullanarak, otoimmün sinir sistemi hastalığı olan etkilenen hastaların bir alt grubu NMDAR yüzey veya D2R yüzey serum antikorları sahip olduğunu bildirmektedir. Canlı hücre bazlı bir deneyde sitometri bu yüksek verimli bir akış...

Son yıllarda, merkezi sinir sistemi (CNS) hastalıkları ve oto-bağışıklık biçimleri tanımlanmıştır. Bu hastalıklar ve ilişkili otoantikorlann mevcudiyeti ile tanımlanır olduğu gösterilmiştir. Bu antikorlar ya da reseptörleri nöronal sinir iletimini 1,2 dahil sinaptik proteinlere bağlanır. Farklı antijenler, örneğin N-metil-D-aspartat reseptör (NMDAR) 3-5, γ-aminobütirik asit reseptör B (GABA B) 6 reseptörü, α-amino-3-hidroksi-5-metil-4-olarak tespit edilmiştir izoksazolepropiyonik asit (AMPA) reseptörü 7, voltaj kapılı potasyum kanal (VGKC) bağlı proteinler: lösin açısından zengin glioma-aktive protein 1 (LGL-1) ve kontaktin-ilişkili protein 2 (capsr2) 8,9, glutamat reseptörü 5,10 ve dopamin-2 reseptörü (D2R) 11. Geçmişte, (çoğunlukla ensefalit olarak da bilinir), bu otoimmün CNS bozuklukları sıklıkla teşhis ve tedavi edilmemiştir. Bu yeni biyolojik antikor, örnNMDAR antikor veya D2R antikor, esasen tanı ve farkındalık düzeldi, ve hastalar için tedavi seçenekleri açtı. Gerçekten de, bağışıklık ile erken tedavi geliştirilmiş sonuç 11,12 ile ilişkilidir. , Örneğin enzim bağlantılı immünosorbent deneyi (ELISA) ve western blot gibi antikorları tespit etmek için geleneksel yöntemler serumunda antikorların saptanması için kullanılmıştır. Ancak, hali hazırda hücre-dışı ya da yüzey epitoplarının tanınması olanak değildir ve, bunun yerine, bir hücre içi epitopuna karşı immunoreaktivitesini ortaya çıkarabilir. Ayrıca, sinir katılan önemli proteinlerin hücre dışı alanına bağlanan antikorlar, patojenik 2,3,7,13,14 olması muhtemeldir. Bu nedenle, ilgili hücre yüzey ya da hücre-dışı hastalarda antikor hedefleri keşfetmek için doğru ve hassas analizlerin geliştirilmesi önemlidir. Alanındaki altın standart yöntem olarak adlandırılan, &quot;hücre-ba olarak, canlı hücrelerin kullanımına dayanmaktadırsed deneyleri &quot;. Bu yöntem, memeli hücrelerinin ilgilenilen antijenin tam cDNA dizisini ihtiva eden vektörler transfeksiyonu ile kendi doğal biçiminde (en çok insan embriyonik böbrek 293 (HEK293) hücre) yüzeyinde bir antijenin ekspresyonunu içerir. Geçirgen hale getirilmemiş canlı hücreler daha sonra, seyreltilmiş hasta serumu ile inkübe edildi ve florokrom-eşlenik anti-insan immünoglobülin (Ig) sekonder antikoru ile takip edilmektedir. Floresans yoğunluğu veya seviyesi, daha sonra tespit edilir ve bağlayıcı otoantikor seviyesine ilişkilidir. Tek bir antijen hücrelerinde aşırı eksprese olduğu gibi bu teknik, özgüdür. En çok &quot;Okuması&quot; kullanılmış immünsitokimya 4,5,8-10 sonra konfokal mikroskopi analizi olmuştur. Bununla birlikte, hücre bazlı deneylerde akış sitometrisi başarılı demiyelinizan hastalıklar 15-17 olan hastalarda antikorları tespit etmek için kullanılmıştır. Özellikle, Waters vd. 15, bir tava ile bir antikorun saptanmasını karşılaştırıldığındaHücre bazlı mikroskobu ile takip deneylerinde ya da dahil olmak üzere, akış antikor algılama teknikleri el sitometri analizleri ve en hassas, doğru ve güvenilir bir yöntem olarak hücre bazlı bir deneyde akış sitometrisi göstermiştir. Bu araştırmacı bağımlı, kantitatif değildir ve radyoaktif malzemenin herhangi bir şekilde kullanılması anlamına gelmez Bu nedenle, hücre bazlı bir deneyde sitometri akışı avantajlıdır. Bu büyük hasta tabur kısa bir miktarda tahlil edilecek verir gibi de uygundur. Daha yakın zamanlarda, otoimmun CNS hastalıkları olan hastalarda 11 NMDAR antikor ve D2R antikoru belirlemek için hücre bazlı bir deneyde sitometri yüksek verimli bir akış optimize ettik. Bizim grubu son hücre bazlı bir deneyde akış sitometrisi kullanılarak hasta serumunda NMDAR antikor tespit edildi. Bu NMDAR antikoru pozitif serumlar daha önce konfokal mikroskopi kullanılarak analiz edildi ve 11 pozitif olduğu bulunmuştur. Bu protokol, c tespiti için hücre bazlı bir deneyde bir akış sitometri özetlenmektedirotomatik yüksek verimli örneği kullanan hasta serumunda onformation duyarlı CNS antikor.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">pIRES2-EGFP vector</td>
			<td style="width:187px;">Clontech</td>
			<td style="width:136px;">6029-1</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Human HA-Dopamine-2 receptor (D2R) cDNA</td>
			<td style="width:187px;">Missouri S&#38;T cDNA Resource Centre</td>
			<td style="width:136px;">DRD020TN00</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Human subunit 1 of NMDAR (NR1) cDNA</td>
			<td style="width:187px;"></td>
			<td style="width:136px;"></td>
			<td style="width:173px;">Gift from Prof A. Vincent&#160; (Oxford, UK)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Monoclonal purified mouse anti-human NR1 antibody (clone: 54.1)</td>
			<td style="width:187px;">BD Pharmingen</td>
			<td style="width:136px;">556308</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Mouse purified monoclonal anti-DRD2 IgG (clone: 1B11)</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:136px;">WH0001813M1-50UG</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Alexa Fluor 647 donkey anti-mouse IgG (H + L)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">A31571</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Alexa Fluor 647 goat anti-human IgG (H + L)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">A21445</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Dulbecco&#8217;s Modified Eagle Medium (1x) 4.5 g/L D-glucose, L-Glutamine and 110 mg/L sodium pyruvate</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">11995-065</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Foetal bovine serum</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">10099-141</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Gentamicin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15710-072</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">GlutaMAX</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">35050-061</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Penicillin-streptomycin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15140-122</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Geneticin</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">10131-035</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:255px;">Dulbecco&#39;s Phosphate-Buffered Saline (1x) (-Ca2+/-Mg2+)</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">14190-144</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">TrypLE Express (1x) with Phenol Red</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">12605-028</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">0.9% Sodium chloride</td>
			<td style="width:187px;">Baxter</td>
			<td style="width:136px;">AHF7975</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Polyethylenimine</td>
			<td style="width:187px;">Polysciences Inc</td>
			<td style="width:136px;">9002-98-6</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Versene</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">15040-066</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">XhoI</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10899194001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">NheI</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10885843001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">PureLink PCR Purification Kit</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:136px;">K3100-01</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">JetQuick Gel Extraction Spin Kit</td>
			<td style="width:187px;">Genomed</td>
			<td style="width:136px;">420050</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">T4 DNA Ligase</td>
			<td style="width:187px;">Roche</td>
			<td style="width:136px;">10481220001</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Plasmid&#160;Plus&#160;Maxi Kit</td>
			<td style="width:187px;">Qiagen</td>
			<td style="width:136px;">12963</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;"></td>
			<td style="width:187px;"></td>
			<td style="width:136px;"></td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">Name of Equipment/Software</td>
			<td style="width:187px;">Company</td>
			<td style="width:136px;">Catalog Number</td>
			<td style="width:173px;">Model/Version</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Flow cytometer with high-throughput sampler system</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">BDLSRII with HTS</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:255px;">Inverted microscope</td>
			<td style="width:187px;">Olympus</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">CKX41 (with mercury lamp and U-RFLT50 power supply)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:255px;">Electronic multichannel pipette (15-300 &#956;l)</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:136px;">613-2240P</td>
			<td style="width:173px;">12-channel Xplorer Plus</td>
		</tr>
		<tr height="56">
			<td height="56" style="height:56px;width:255px;">Excel</td>
			<td style="width:187px;">Microsoft</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Prism</td>
			<td style="width:187px;">GraphPad Software, Inc.</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">v4</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:255px;">FlowJo</td>
			<td style="width:187px;">Treestar</td>
			<td style="width:136px;"></td>
			<td style="width:173px;">v7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">50 ml polypropylene conical tubes</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">352070</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">6-well plate</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">353046</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Tissue culture flask (T75)</td>
			<td style="width:187px;">BD Biosciences</td>
			<td style="width:136px;">353136</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:255px;">Parafilm</td>
			<td style="width:187px;">Pechiney Plastic Packaging</td>
			<td style="width:136px;">PM-992</td>
			<td style="width:173px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:255px;">V-bottom 96-well plate</td>
			<td style="width:187px;">Corning</td>
			<td style="width:136px;">651180</td>
			<td style="width:173px;"></td>
		</tr>
	</tbody>
</table>

high-throughput flow cytometry cell-based assay to detect antibodies to n-methyl-d-aspartate receptor or dopamine-2 receptor in human serum

Son yıllarda, yüzey ve sinir iletimi ile ilgili yapısal proteinlerine karşı olan antikorlar, çocuklar ve yetişkinler için CNS otoimmün hastalıklarda tespit edilmiştir. Bu antikorlar tanı ve tedaviye yol kullanılmıştır. Hücre bazlı deneyler hasta serum içinde antikorların saptanmasını iyileştirilmiştir. Daha sonra florokrom-konjuge sekonder antikor ile, ardından seyreltilmiş hasta serası ile inkübe edilir, ökaryotik hücreler üzerinde beyin antijenlerin, yüzey ekspresyonu dayanmaktadır. Yıkama işleminden sonra, bağlayıcı ikinci antikor daha sonra akış sitometrisi ile analiz edilir. Bizim grup güvenilir bir şekilde belirli bir nörotransmiter reseptörlerine karşı antikorlar tespit etmek için, canlı hücre bazlı bir deneyde sitometri yüksek verimli bir akış geliştirmiştir. Bu akış sitometri yöntemi yalındır, kantitatif, verimli ve geniş hasta kohortlarının kolayca zaman kısa bir alanda test edilmesi için yüksek verimli bir örnekleyici sistemi kullanma imkanı sağlar. Ayrıca, bu gibi hücre bazlıki kolayca merkezi sinir sistemi ve çevre hem de birçok farklı antijenik hedeflere antikorları tespit etmek için adapte edilebilir. Ilave yeni antikor biomarkerlerin Keşfi hızlı ve doğru tanı sağlamak ve bağışıklık aracılı bozuklukların tedavisini artıracaktır.

Canlı HEK293 D2R + ve HEK293 hücreleri, CTL yüksek verimli bir örneği kullanarak akış sitometresi ile elde edildi. Analiz sırasında, hücreler ileri dağılım (boyut) ve yan dağılım (granülaritesi) parametreleri (Şekil 1A ve 1B) dayalı kapılı edilmiştir. Transfekte edilmiş HEK293 hücreleri sitoplazmasında raportör molekülü, GFP ifade ve transfekte edilmemiş hücrelerin analizi (Şekil 1 B ve 1 E) alınmad?...

Watch this Scientific Journal Video about High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum at JoVE.com

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Son yıllarda, canlı hücre bazlı deneyler, yüzey ve şekilsel antijenlere karşı antikorlar tespit etmek için başarılı bir şekilde kullanılmıştır. Burada, hastaların büyük kuşaklarının analiz sağlayarak sitometri yüksek verimli akışını kullanarak bir yöntem açıklanmaktadır. Yeni antikorların tespiti immün aracılı bozuklukların tanı ve tedavi artıracaktır.

İnsan Serum içinde N-metil-D-aspartat reseptör ya da dopamin-2 reseptör antikorları algılamak üzere yüksek verimli Flow Cytometry hücre bazlı bir deneyde

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

high-throughput-flow-cytometry-cell-based-assay-to-detect-antibodies

Research

JoVE Journal

Medicine

16.0K Görüntüleme.  The University of Sydney.  Son yıllarda, canlı hücre bazlı deneyler, yüzey ve şekilsel antijenlere karşı antikorlar tespit etmek için başarılı bir şekilde kullanılmıştır. Burada, hastaların büyük kuşaklarının analiz sağlayarak sitometri yüksek verimli akışını kullanarak bir yöntem açıklanmaktadır. Yeni antikorların tespiti immün aracılı bozuklukların tanı ve tedavi artıracaktır. 

Video: High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

Ambliyopi içinde Bastırma Ölçme ve Tedavisi

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Tıp

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Geçici veya kalıcı Serebral İskemi Modelinde lökosit alt popülasyonlarının Stereolojik ve Sitometrisi Karakterizasyonu

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

İnsan Videofloroskopik Kırlangıç ​​Eğitim Yöntemleri uyarlanması Algılama ve Mürin Hastalık Modelleri disfajiyi Characterize

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

Meme Kanserinde Östrojen Reseptör Sinyali tarafından Metabolik Yönetmelik Sistem Biyolojisi

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

A High Throughput, Multiplexed and Targeted Proteomic CSF Assay to Quantify Neurodegenerative Biomarkers and Apolipoprotein E Isoforms Status

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be important in the development of future novel therapies. Often&#160;potential new biomarkers do not make it&#160;into the clinic&#160;due to limitations&#160;in their development and&#160;high costs.&#160;However,&#160;targeted proteomics using Multiple Reaction Monitoring Liquid Chromatography-tandem/Mass Spectrometry (MRM LC-MS/MS), specifically using triple quadrupole mass spectrometers, is one method that can be used to rapidly evaluate and validate biomarkers&#160;for clinical translation into diagnostic laboratories. Traditionally, this platform has been used extensively for measurement of small molecules&#160;in clinical laboratories, but it is the potential to analyze proteins, that makes it an attractive alternative to ELISA (Enzyme-Linked Immunosorbent Assay)-based methods. We describe here how targeted proteomics can be used to measure multiplexed markers of dementia, including the detection and quantitation of the known risk factor apolipoprotein E isoform 4 (ApoE4).
In order to make the assay suitable for translation, it is designed to be rapid, simple, highly specific and cost effective. To achieve this, every step in the development of the assay must be optimized for the individual proteins and tissues they are analyzed in. This method describes a typical workflow including various tips and tricks to developing a targeted proteomics MRM LC-MS/MS for translation.
The method development is optimized using custom synthesized versions of tryptic quantotypic peptides, which calibrate&#160;the MS for detection and then spiked into CSF to determine correct identification of the endogenous peptide in the chromatographic separation prior to analysis in the MS. To achieve absolute quantitation, stable isotope-labeled internal standard versions of the peptides with short amino acid sequence tags and containing a trypsin cleavage site, are included in the assay.

We describe a high-throughput, multiplex, and targeted proteomic cerebrospinal fluid (CSF) assay developed with potential for clinical translation. The test can quantitate potential markers and risk factors for neurodegeneration, such as the apolipoprotein E variants (E2, E3 and E4), and measure their allelic expression.

Bir Yüksek Verimli, Çoklanmış ve Hedefli Proteomik BOS Deneyi nörodejeneratif Biyomarkerleri ve Apolipoprotein E İzoformları Durum belirlememize

Many neurodegenerative diseases are&#160;still lacking effective treatments. Reliable biomarkers for identifying and classifying these diseases will be ...

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

Bir Kinetik Floresans tabanlı Ca2 + seferberlik tahlil G Protein birleştiğinde reseptör agonistleri, antagonistleri ve Allosteric modülatörler tanımlamak için

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Akış Sitometresi kullanarak insan yağ dokusu içinde bağışıklık hücreleri karakterizasyonu

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Kardiyak miyositler mitokondrial membran potansiyeli hipoksi/Reoxygenation sonra ölçmek için akış sitometresi tabanlı tahlil

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Measurement of in vitro cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool as a biomarker for atherosclerosis. In the present study, we optimize and standardize a fluorescent NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Yüksek işlem hacmi Nitrobenzoxadiazole etiketli kolesterol sızma tahlil

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

Biochip mikrosıvısal teknolojisi uygulama algılamak Serum alerjen özgü immünglobulin e (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...