Yazarlar ölçüde Pierre BOURDONCLE, Thomas Guilbert ve Cochin Görüntüleme Tesisi Louise Rimbault kabul. Bu çalışma Inserm, CNRS ve Ligue Nationale contre le Kanser (Equipe labellisée) tarafından desteklenmiştir.

T Lemfositleri hazırlanması 1. <ol><li> 10,11 tarif edildiği gibi primer, fare veya insan T hücrelerini hazırlamak. </li><li> 3 x 10 6 hücre / mL - 2 arasında bir yoğunlukta% 10 insan serumu AB ile tam RPMI ortamı içinde insan T hücrelerini yetiştirmek. Not: kültürde insan T hücrelerinin büyük bir kısmını kendiliğinden polarizasyon ortaya fetal sığır serumu (FCS) yerine, insan serumu kullanılması. Bu dönemde herhangi bir zamanda 5 gün ve daha fazla işlem onları - 4 kültürde bu hücreleri tutun. </li><li> Benzer bir hücre yoğunluğunda,% 10 FCS ile desteklenmiş tam RPMI ortamı içinde fare T hücrelerini koruyun. 10 ng / ml IL7 mevcudiyetinde 37 ° C &#39;de hemen ya da ertesi gün, bir O / N kültürden sonra kullanın. </li><li> Tam RPMI,% 10 FCS ile takviye edilmiş CEM insan T hücre soyu geliştirin. </li></ol> 2. Kemokin Uyarım <ol><li> 10 mM HIPE ile desteklenmiş sıcak HBSS ortamında deneysel koşul başına 5 x 10 5 hücre yıkayıns. Bazı deneyler için, 2 ug pmaxGFP ve 8 ug pcDNA3.1 + (boş vektör kontrol), ölü yer PKCζ kinaz ya da N-ucu Par6 plazmidler ile bir gün önce, primer insan T hücrelerinin ko-transfekte. </li><li> Sıcak HBSS-hepes ortamında hücre pelletini (deneysel koşullar 0.3 ml x numarasını kullanın). </li><li> 1.5 ml mikrosantrifüj tüplerine hücreleri dağıtın. NOT: Bu nedenle, tek bir deneysel koşuluna karşılık gelen bir boru, 0.3 ml HBSS-HEPES ortamında 5 x 10 5 hücre içerir. Bazı deneyler için, 500 nM Latrunculin A veya aracı (DMSO) ilave ediniz ve kemokin uyarılmadan önce 30 dakika için kuluçkalayın. </li><li> Her bir tüp içinde 10 ng / ml CCL19 veya CXCL12 kemokin 500 ekleyin. NOT: Biz genellikle 10 kullanın - İnsan T hücreleri için 200 ng / ml kemokini. CEM hücreleri CCL19 bağlayan CCR7 reseptörü ifade yoktur. Bu nedenle, CEM hücreleri sadece bir CXCL12 uyarısına cevap mümkün olacak. 500 N - Ayrıca, daha yüksek bir kemokin konsantrasyonları (300 kullanımıg / ml) fare T hücrelerini kutuplaşmaya. </li><li> Tüpler birkaç kez ters çevirin ve sırasıyla, birincil T hücrelerinin veya CEM 8 ya da 10 dakika boyunca 37 ° C su banyosu içinde inkübe. </li><li> Her bir tüpe,% 2 paraformaldehit (PFA) ile PBS içinde 10 mM Hepes ihtiva eden ılık bir çözeltiye, 0.3 ml ilave etmek suretiyle polarizasyon işlemi durdurun. Tüpler ters çevirin ve 5 dakika boyunca 37 ° C&#39;de inkübe. </li></ol> 3. Renklendirmeler <ol><li> Tüpler akış sitometri mikrosantrifüj tüpleri hücreleri aktarın. </li><li> % 1 sığır serum albümini (BSA), 0.5 mM EDTA ve PBS içinde 10 mM Hepes ihtiva eden bir RT çözeltisi ile tam olarak tüpler doldurun. </li><li> 4 dakika için 460 xg&#39;de tüpler santrifüj. </li><li> Süpernatant atılır ve PBS içinde% 5 FCS ihtiva eden bir RT çözeltisi 100 ul süspansiyon hücrelerin. </li><li> Işıktan uzakta, girdap onları, her bir tüp içinde FITC-anti-HLA-ABC 5 ul ekleyin ve oda sıcaklığında 30 dakika boyunca inkübe. </li><li> PBS,% 5 FCS ihtiva eden bir kez hücreler yıkayınSadece bir kez PBS ile nd. </li><li> Oda sıcaklığında: daha sonra% 1 BSA, 0.5 mM EDTA ve PBS içinde 10 mM HEPES içeren bir solüsyon ile yıkama hücreleri, 10 dakika süre ile inkübe edilir, 500 ul% 1 PFA ekleyin. </li><li> Süpernatant atılır, PBS,% 0.2 BSA,% 0.1 saponin, 0.5 mM EDTA ve 10 mM Hepes (permeabilizasyon tamponu) ihtiva eden bir çözelti ile tamamen tüpleri doldurun. </li><li> Bu ortam içinde hücreler iki kez yıkayın. </li><li> Orta kaldırmak ve hücre pelet ayırmak için onları vorteks tüpleri çevirin. </li><li> Her bir tüpe, anti-P-KRY antikorun 100 seyrelti, girdap onları ve oda sıcaklığında 30 dakika inkübe edilir: ve / veya 1 ila 0.25 ug / ml TRITC-falloidin ekleyin. </li><li> Permeabilizasyon tampon maddesi ile hücreleri yıkayın. Bir fluorescent sekonder antikor gerektiğinde kuluçkalayın. </li><li> 200 ul PBS hücrelerin tekrar ve mikrosantrifüj tüpler aktarabilirsiniz. NOT: Hücreler edinimi için hazırız. Tüp başına 200 ul maksimum toplam hacmi korurken, alternatif olarak, bir son% 1 konsantre PFA eklemekHücreler daha fazla işlem için aynı gün kullanılmak üzere takdirde için yoğunlaştırma lekelenmelerinin korumak için. </li></ol> 4. Görüntüler Toplama ve Analiz <ol><li> Aparat (Belirli Reaktifler / Ekipman Tabloya bakınız) açın ve üreticinin talimatlarına göre kendisini kalibre edelim. NOT: INSPIRE yazılımı 40X objektif (0.75 NA) ile kullanılmıştır. FİKİRLER 3.0 yazılımı tüm bildirilen sayımsal için kullanılmıştır. </li><li> Gelecek deneyler için bir şablona kaydedilebilir satın alma pencereleri bir dizi hazırlayın. RMS degrade değerlerini ölçmek histogramlar çizin. RMS degrade histogram seçilen &quot;Focus&quot; nüfus itibaren, Alan histogram &quot;Tek hücreli&quot; nüfus sınırlandırmak. Mikrosantrifüj tüpü makinesinde yerleştirildikten sonra, tek tek hücreler ile bir kapı lazer gücünün ayarlanması ve 5000 T lenfositleri kazanır. </li><li> Yumuşak Fikirlerware, satın alma dosyasını açın ve her hücre için elde edilen parlak bir alan görüntüler için degrade RMS değerini gösteren bir histogram (Kanal 1) oluşturun. 57 Yukarıda RMS degrade değeri sergileyen hücreler gördüğü RMS degrade histogram üzerinde bir kapı çizin. NOT: RMS degrade analizinde odak düzleminde sadece T lenfositler dikkate alarak sağlar. Bu 57 um&#39;nin RMS gradyan değeri gösteren tek tek hücreler odak düzlem içindedir ve doğru olarak kabul edilebilir ampirik olarak tespit edilmiştir. </li><li> Analiz / Maskeler menüsünde, 2 piksel aşınmış aydınlık görüntüler M01, üzerinde morfoloji maskesinden (M01,2) Erode denilen, yeni bir maske oluşturmak. Ardından, Analiz / Erode (M01,2) maske hesaplanan Area, yeni bir özellik oluşturmak, menü bulunmaktadır. Bir önceki kapısı seçilen hücrelerden, bu yeni oluşturulan maske kullanarak hücrelerin Alan gösteren bir histogram açın. NOT: ulaşırsınız morfoloji maskeleriVarsayılan hücrenin gerçek boyutundan daha büyüktür. Böylece, 2 piksel o kadar aşındırmak için daha hassas. </li><li> Kalibrasyon boncuk ve enkaz (küçük Alan) ve çiftler (büyük Area) hariç, bireysel T hücreleri üzerinde bir kapı çizin. Her satın alma bu kapıyı ayarlayın. NOT: Hücre boyutu varyasyonlar kapısı konumunu etkileyecektir. Fare T hücrelerinin kendilerini CEM hücreleri daha küçük olan insan T hücrelerinin daha küçük olduğu için, her hücre tipi alan büyüklüğü ile orantılı olacaktır. </li><li> Önceki adımlarda Geçitli edildi tek odakta hücreleri göz önüne alındığında, Phalloidin Raw Max Pixel bir fonksiyonu olarak HLA-ABC boyama Raw Max Pixel (Kanal 2, x ekseni) oluşan bir nokta arsa açmak boyama (Kanal 4, y ekseni). Lekesiz veya doymuş floresan olayları dışlamak için bir kapı oluşturun. Ham Max HLA-ABC için Pixel (Kanal 2) ve Phalloidin (Kanal 4) lekelenmelerinin gösteren Aç iki histogramlar. </li><li> Analiz / Maskeler menüsünde, creat içinde2 piksel aşınmış HLA-ABC boyanmış hücreler (Kanal 2), morfolojisi maskesinden (M02,2) Erode denilen adet yeni maske. Ardından, Analiz / Erode (M02,2) üzerinden hesaplanan Circularity parametresi oluşan yeni bir özellik oluşturmak, menü bulunmaktadır. NOT: Bu parametre, bir daire hücre şeklinin sapmasını ölçer. Bu nedenle, mükemmel yuvarlak T hücresi düşük dairesellik endeksi sergileyecek uzatılmış polarize hücreler ise yüksek bir yuvarlaklık değerine sahip olacaktır. </li><li> Daha sonra, daha önce kapı hücrelerden Circularity özelliği değerlerini rapor başka bir histogram yaratmak. Doğrudan her bir hücre için daireselliğe değerine göre bir birey, odakta hücre popülasyonu dağılımının çizmek için bu histogram kullanın. </li><li> Uyarılmamış hücrelere için histogram şeklinde bakıldığında, dairesellik sınır değerine kadar düşük dairesellik değerden başlayarak polarize hücreler için bir kapı çizmek burada Uyarılmamış hücrelere ençizilmiştir. Kapılı nüfus arasında polarize hücrelerin yüzdesi istatistikleri ekrana bak (% Geçitli). NOT: Biz 13 altında Circularity indeks değerleri için bu kapıyı belirledik. </li><li> Hücrelerde aktin boyama kutuplaşma bakmak için, HLA-ABC boyama (Kanal 2) ve falloidin boyama karşılaştırarak, Parlak Detay Benzerlik R3 değeri için histogram arsa (Kanal 4). NOT: Bu özellik büyük değeri, en fazla iki Renklendirmeler vardır bindirilmiş. </li><li> Daha önce olduğu gibi aynı yolluk strateji kullanarak, polarize aktin boyama ile hücrelerin yüzdesini gösterir ayrılmış boyama için, uyarılmamış hücreler ile bir kapı çizilir. </li><li> Tamamlandığında, MHC Sınıf I ve falloidin boyamalar, birlikte SD ± bütün hücrelerin Circularity ve Benzerlik indeksleri ortalama değerleri ile polarize hücrelerin yüzdesi dağılımında bir ayrımı sergileyen hücrelerin yüzdesi Nominal değerden uygun olarak gösterilmektedironding istatistik paneller. </li></ol>

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Yazarlar hiçbir rakip mali çıkarlarını olduğunu beyan ederim.

Sitometri, hızlı ve bilgilendirici yolluk strateji kemokin uyarımı ile uyarılan hücresel ve moleküler olayları analiz etmek görüntüleme akışının son teknolojiyi kullanarak sunulmuştur. Kemokin uyarılması ve polarizasyon sürecinde farklı proteinlerin hücre içi dağılımı ile oluşturulan hücre morfolojisi değişiklikleri: Tek bir deney itibaren, bir iki ana bilgi edinebilirsiniz. İlginç bir şekilde, kanıtlar da istatistiksel sağlamlık ve bütün hücre nüfusun küçük bir fraksiyonu ilgi...

Kemokinler özel yerlere 1 hücreleri çekmek küçük çözünebilir proteinlerdir. Bu nedenle, bu dokularda hücre doğru konumlandırılması, geliştirme ve fizyolojisi çok önemli bir işlevine katılırlar. Bu, etkili bir bağışıklık karşılığının ortaya çıkarılması için birlikte hareket birçok farklı hücre tiplerinin hareketlerine dayanır, bağışıklık sistemi, bu kurala istisna değildir. Yabancı antijenler tespit ve nötralize edilebilir önce belirli bir durumda bir bağışıklık hücre tipi özel konumunu kontrol ederek, kemokinler önceden gerekli bulunmaktadır. Özellikle T lenfositleri kemokinler T hücresi hareketliliği 2,3 tercih geçişi üzerine, hücreiçi sinyal ortaya, özel yüzey reseptörleri (kalsiyum yükselişi ERK fosforilasyonu, Rho GTPazlar aktivasyonu, integrinler afinite ve sitoskeletal değişimler artış) bağlanır. Hücresel düzeyde, bir kemokin stimülasyon üzerine ortaya morfolojik değişiklikler gözlemleyebilirsiniz.Hücre şekilleri bu değişiklikler T hücrelerinin özellikle dramatik: kanda seyahat ederken dinlenme T hücreleri, bir boncuk gibi yuvarlak morfoloji var. Ön bir ön kenarı: Bununla birlikte, iltihabik bölgelerde veya lenfoid organların yakınlık kemokinlerin varlığı algılama hemen iki kutuplu bir formundan oluşan tipik bir &quot;el ayna&quot; morfolojisi kabul T hücrelerinin şeklinin değiştirilmesi için gidiyor ve arka 4 bir arka kenar veya uropod. Buna ek olarak, hücre içi bileşenleri göç sürdürmek için polarize T hücresinin bu iki zıt bölgeye ayırmak olabilir. Örneğin kemokin uyarımı 5 ve polimerize aktin üzerine aktin filamentler polimerizasyon artar kutuplaşmış T hücresinin 2 ön birikir. Öte yandan, bu tür kortikal F-aktin hücre iskeleti, plazma membranı bağlantı Ezrin-Radixin-moesin (ERM); ailesinin fosforile proteinler gibi bazı proteinler, kutuplaşmış uropod yeniden lokalizeT hücreleri, 6. İlginç bir şekilde, ve diğerleri, bu kutuplaşma süreci T hücre göçü için gerekli olduğunu göstermiştir. Nitekim, polarizasyon müdahale herhangi bir tedavi hücresel hareketliliğini engeller. Örneğin, atipik protein üyelerinin etkinliğinin engellenmesi, C (PKC), aile, PKCζ ve PKCι blok T hücresi polarizasyon ve dendritik hücrelerin 7 için göç tarama işlemini kinaz. T hücre kutuplaşma da Rho GTPases tarafından düzenlenir. Son zamanlarda tarif edildiği Fam65b proteini ile RhoA aktivitesinin modüle bir Transwell tahlilinde 6 geçirilecek T hücre morfolojisindeki değişikliklerle ve kapasiteyi etkiler göstermiştir. Polarizasyon hücre motilitesi için bir ön adım olarak, kemokin tepkilerin ana okuma olarak ölçmek mümkün böylece önemlidir. Hücre şekli değişiklikleri önceden elle 8 ölçüldü. Ancak, miktar bu tür çok zaman tüketen olduğunu, genellikle sadece birkaç yüzdenhücre on dikkate alınmaktadır. Burada, yeni bir yöntem hızlı uyarımı kemokin maruz bırakılan T lenfositlerinin şekil değişiklikleri derecesini ölçmek için sunulmuştur. Teknoloji sitometri bir görüntüleme akış kemokin stimülasyon farklı koşullarda verimli polarize hücrelerin miktarını ölçmek için bir akış sitometresinde ve bir mikroskop 9 avantajlarını birleştiren, kullanılan (Belirli Reaktifler / Ekipman Tablo). Bir bu teknoloji ile sağlam ölçebilen morfolojik değişimlerin değerlendirilmesiyle ek olarak, bu kemokin sinyali üzerine, bazı proteinlerin hücre altı lokalizasyonda değişiklikleri değerlendirmek için de mümkündür.

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			<td height="43" style="height:43px;width:140px;">Name of the material/Equipment</td>
			<td style="width:95px;">Company</td>
			<td style="width:80px;">Catalog number</td>
			<td style="width:255px;">Comments/Description (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">RPMI</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">61870-010</td>
			<td style="width:255px;"></td>
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		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Human serum AB</td>
			<td style="width:95px;">PAA</td>
			<td style="width:80px;">C11-021</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
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		<tr height="37">
			<td height="37" style="height:37px;width:140px;">Fetal calf serum</td>
			<td style="width:95px;">PAN Biotech</td>
			<td style="width:80px;">P30-3300</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:140px;">Human T cell nucleofactor kit</td>
			<td style="width:95px;">Lonza</td>
			<td style="width:80px;">VCA-1002</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Murine IL7</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">217-17</td>
			<td style="width:255px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:140px;">HBSS</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">14025-050</td>
			<td style="width:255px;">Warm in 37 &#176;C water bath before use.</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Hepes</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">15630-056</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:140px;">Murine CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">250-27B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:140px;">Human CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-29B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:140px;">Human CXCL12</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-28A</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:140px;">PFA</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:80px;">157-8-100</td>
			<td style="width:255px;">This is a 8 % PFA solution in water. Mix volume to volume with 2X PBS to obtain a 4 % PFA solution in PBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">&#160;BSA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:80px;">A3059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Saponin</td>
			<td style="width:95px;">Fluka</td>
			<td style="width:80px;">84510</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Alexa Fluor 594 phalloidin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:80px;">A12381</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">FITC-anti-HLA-ABC antibody</td>
			<td style="width:95px;">Beckman Coulter</td>
			<td style="width:80px;">IM1838</td>
			<td style="width:255px;">clone B9.12.1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Anti-P-ERM antibody</td>
			<td style="width:95px;">Cell Signaling Technology</td>
			<td style="width:80px;">3149P</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:140px;">ImageStreamx Mark II</td>
			<td style="width:95px;">Amnis</td>
			<td style="width:80px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling

Hücreler, bir süreç olarak adlandırılan kutuplaşma onların yuvarlak kaybederek ve birçok proteinlerin hücre içi lokalizasyonu değiştirerek uyarılmasını kemokin cevap. Klasik görüntüleme teknikleri bu olguları incelemek için kullanılmıştır. Bununla birlikte, morfolojisi ve hücre onlarca boyama eş-lokalizasyonu zaman alıcı miktarının ardından birçok hücrede elle toplama gerektiriyordu. Burada, hızlı ve güçlü bir yöntem, bir sitometresinde kişilerce mikroskop avantajlarını bir araya teknolojisi sitometri bir görüntüleme akışı kullanılarak hücrelerin birkaç bin oluşan numuneler üzerinde, bu olayları incelemek için tarif edilmektedir. MHC sınıf I molekülleri ve filamentli aktin için CCL19 ve boyama ile uyarılmış T lenfositleri kullanılarak bir yolluk stratejisi aynı zamanda şekil değişikliklerinin derecesi ve CCL19 sinyal etkilenen belirteçlerin eş-lokalizasyonu derecesini ölçmek için sunulmuştur. Üstelik, bu yolluk strateji gözlemleme için bize izine CXCL12 uyarıldıktan sonra polarize T hücrelerinde (ön) ipliksi aktin ve (arkada) fosforile Ezrin-Radixin-moesin (fosfo-ERM) proteinlerin ayrımı. Par6 / atipik PKC sinyal yolunun mutantlar bir farmakolojik inhibitörü ve ifade ile aktin polimerizasyonu inhibe: Bu teknik aynı zamanda iki farklı unsurların kutuplaşma engelleme etkisini gözlemlemek için yararlı oldu. Böylece, kanıtlar bu tekniğin morfolojik değişiklikler ve protein yeniden dağıtımı esnasında hem de analiz etmek yararlı olduğu gösterilmiştir.

Burada seçilen ilk örnek CCL19 ile uyarılmış insan primer T lenfositlerinin kullanımı ile ilgilidir. Bununla birlikte, aynı strateji primer fare T hücreleri, T hücre çizgileri ya da kemokin stimülasyona yanıt veren herhangi bir başka hücre tipi ile kullanılabilir. Burada sunulan yolluk strateji odaklı olaylar, ardından tek hücreleri seçin pencerelerin bir dizi içerir. (Şekil 1) veya CCL19-uyarılmış (Şekil 2), T lenfositleri istirahat için MHC Sınıf I HLA-ABC...

Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Kemokin Sinyalizasyonu Bağlı Hücresel ve Moleküler Polarizasyon Hızlı ve Sağlam Analizi

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

rapid-robust-analysis-cellular-molecular-polarization-induced

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Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

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8.0K Görüntüleme.  Institut Cochin. Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Video: Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

Görselleştirme ve Miktar hücre içi Etkileşimleri Neisseria meningitidis</emKonfokal Görüntüleme α-aktinin> ve İnsan

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

İmmünoloji ve Enfeksiyon

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

Damla Akış ve Döner Disk Reaktörler kullanın Staphylococcus aureus Biyofilm Analizi

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

Sivrisinek Fonksiyonel Analiz için Kalsiyum Biyoparlaklık Testi ( Aedes aegypti) Ve Tick ( Rhipicephalus Microplus) G Protein-çiftli Reseptörler

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Görüntülemede HIV-1 Zarf bağlı Virolojik Synapse ve Sentetik Lipid Bilayers üzerine Sinyal

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

Gerçek zamanlı, TCR Eşzamanlı Görüntüleme ve İlişkili Sinyal Proteinler için TIRF Mikroskopi Tekniği

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

Yapımcı Epitel Hasar Analizi<em&gt; Entamoeba histolytica</em&gt; Enfeksiyon

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH ile Fare B Lenfositlerindeki kromozom anormallikleri hızlı Analizi

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Hızlı moleküler algılama ve Nezle Virüsleri A ve B farklılaşması

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Bimolecular tamamlayıcı benzeşme arıtma (BiCAP) tarafından parçalanmış çok protein sinyal kompleksleri

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.