Biz 37 ° C PFA fiksasyon denemek için öneri içeren yararlı tartışmalar için Dr Harold Dvorak kabul. Bu çalışma NIH hibe P01 CA92644 tarafından ve Kanser Araştırmaları Ulusal Vakfı bir sözleşme ile desteklenmiştir.

1.. Kollajen Kaplamalı Camlar Diskte Hücre Kültürü <ol><li> Bir cam kavanoz (4 oz) ve diskler sterilize etmek otoklav içinde yer cam disk (çapı 12 mm). </li><li> Bir 50 ml Falcon tüpü içinde 25 ml% 70 etanol koyun ve bir hücre kültürü kaputu yerleştirin. Çözeltinin seviyesi 20 ml düşene kadar bu çözelti, birden çok kez tekrar edilebilir. </li><li> Cam disk kolu için kullanmadan önce, 5 dakika boyunca% 70 etanol içinde bir ekstra ince nokta ile keskin bir forseps yerleştirin. </li><li> Dikkatlice tüp dışında forseps kaldırmak ve kapağını kapatın, daha sonra yavaşça 2 dakika boyunca hava ile kuruması için tüpün üstüne etanol tedavi forseps yerleştirin. Forseps ucu kaputu şey dokunmak izin vermeyin. </li><li> Cam kavanoz dışında bir steril cam diski kapmak ve 24-yuvalı hücre kültürü plakasının bir kuyu içine yerleştirmek için steril forseps kullanın. Kuyu istenen sayıda diskler ile doldurulan kadar tekrarlayın. </li><li> Bovine kolajen çözeltisi 500 ul (koyun50 ng / ml) bir cam disk içeren ve en az 30 dakika boyunca 37 ° C,% 5 CO2, hücre kültürü yetiştirme cihazı yerleştirmek her kuyuya. Daha uzun bir inkübasyon hiçbir zararı yoktur. </li><li> Hasat HUVEC (insan göbek damar endothelial hücreleri) ya da daha önce PC3 tripsinizasyon ile 150 mm hücre kültürü plakasında yetişen ve tam kültür ortamı kullanılarak tripsin aktivitesini bloke edilmiştir (prostat tümör hücreleri). Bir 15 ml Falcon tüpü içine hücreleri toplamak. </li><li> Hücre sayısını ve canlılığı% 90 daha büyük olduğundan emin olun. </li><li> 4 ° C&#39;de 10 dakika boyunca 200 x g santrifüj ile hücreler Pelet Süpernatant kaldırmak. </li><li> 10 5 hücre / ml olacak şekilde seyreltmek hücreleri için kültür ortamı kullanın. Hücreleri buz üzerinde yerleştirin. </li><li> Hücre kültürü kaputu kolajen ve yavaşça aspire cam diskler kolajen ile kaplanmış edildiği de her bir hücre süspansiyonu, 500 ul koyun. 24 oyuklu bir plaka içerisinde, 5 x 10 4 hücre / çukur% 60 conf verecekHUVEC ve PC3 hücreleri için% 30 akma luency. Not: yüksek hücre yoğunluğu için daha fazla hücre süspansiyonu ekleyin. </li><li> Kültür, 37 ° C, 1 saat, 2 saat, 4 saat, 6 saat, ya da nanopodia faaliyetleri ve hücre geçişi izlemek için 24 saat boyunca% 5 CO2 inkübatörü içinde hücreler. Tipik olarak, hücreler ilk 30 dakika içinde cam disk eklemek, daha sonra Polarize ve ilk bir saat içinde göç başlar ve ilerleyen saatlerde arası etkileşimleri ve hücre bölünmesini gerçekleştirin. </li></ol> 2. Hücre Fixation <ol><li> Her iyi hücrelerini düzeltmek için 500 ul% 4 PFA (paraformaldehid) ihtiyacınız olacak. Ticari olarak üretilen ya da taze% 4 PFA kullanılabilir yapılmış ya. Hazırlanan kuyu sayısına göre gerekli PFA miktarını hesaplayın ve bir 15 ml Falcon tüpüne PFA yerleştirin. DİKKAT: paraformaldehıt bir kanserojen olabilir. </li><li> 37 ° C inkübatör yerinde zaten bir strafor stand şahin tüp koymak. Savaş için 30 dakika boyunca inkübatör tüpü terk37 ° C&#39;ye kadar PFA m </li><li> Kültürü kaputu bir ısıtma pedi yerleştirin ve açın. </li><li> 24-iyi hücre kültürü plakası ve kuluçka prewarmed PFA çıkarın ve ısıtma yastığı üstüne yerleştirin. </li><li> Bir kuyudan orta aspire bir elinizi kullanın ve hemen sonra hafifçe iyi kenarı boyunca kuyuya 500 ul PFA slayt için diğer elinizi kullanın. Kolay aspirasyonu için, o açıyla istikrarlı olduğunu böylece Strafor standı dayayarak, yaklaşık 45 ° de kültür plaka eğin. Bu, aspirasyon ile kültür içinde hücreleri bozmadan oyuğuna PFA çözeltisi eklenerek kolaylaştıracaktır. Bu hücre faaliyetleri, orijinal hücre yapısını korumak için en önemli adımdır. </li><li> Bir cam disk ile tüm kuyuları PFA almış kadar işlemi tekrarlayın. Not: kuyudan önceden varolan çözüm değiştirmek için gereken tüm adımlar aynı aspirasyon prosedürleri uygulayacaktır. </li><li> 5 dakika boyunca 37 ° C &#39;de plaka koyun. </li&gt; <li> Geri kültürü kaputu plaka almak ve adım 2.5 açıklandığı gibi Strafor karşı yatırın. Nazikçe bir toplama tüpüne kuyudan PFA aktarmak için bir elinizi kullanın; hemen sonra da en az 500 ul oda sıcaklığında PBS yerleştirmek için diğer elinizi kullanın. Tüm çukurlar yıkanıp sonra, geri ve de her bir kere daha PBS yıkama tekrarlayın. Tehlikeli atık kabı içine toplanır PFA boşaltın. </li><li> PBS,% 2 fetal sığır serumu eklenerek ICC bloke tamponu hazırlayın. % 0.04 sodyum azid (% 20 stok çözeltisinden seyreltilmiş), bir koruyucu madde olarak ilave edilir. DİKKAT: Sodyum azid zehirli bir bileşiktir. Tampon bakteriyel kontaminasyon olmaksızın uzun bir süre için 4 ° C&#39;de saklanabilir. </li><li> Yine de her iyi PBS kaldırmak için aspire ve hemen sonrasında ICC engelleme tampon 500 ul ekleyerek aracılığıyla döngüsü bir kez, bir stand karşı eğik kültür plaka ile. Hücreler artık immunofloresan boyama için hazır bulunmaktadır. Gerekirse,Sabit hücre TM4SF1 proteinin önemli bir kayıp olmaksızın bir hafta boyunca 4 ° C&#39;de buzdolabında saklanır. </li></ol> 3. Nanopodia arasında TM4SF1 İmmünofloresan boyanması <ol><li> Her bir oyuğa olarak, Triton X-100% 0.01 ihtiva eden yeni bir bloke edici tampon ile 500 ul bloke edici tampon ICC değiştirmek ve 1 saat boyunca oda sıcaklığında bekletin. Hücre zarlarından TM4SF1 kaldırmak gibi% 0.03 Triton X-100 daha yüksek kullanmayınız. Boyama hemen başlamak için hazır olup olmadığını PBS adım 2.10 ile yıkanmış hücrelerini doğrudan Triton içeren engelleme tampon taşınmış olabilir. </li><li> ICC bloke etme tampon maddesi içinde 0.5 ug / ml anti-birincil TM4SF1 (ya da anti-CD9) antikoru seyreltin. </li><li> Her bir oyuğa olarak, ICC/0.01% Triton tamponunu çıkarın ve anti-TM4SF1-antikor çözeltisi 300 ul ile değiştirin. Oda sıcaklığında 2 saat inkübe ya da 4 ° C&#39;de bir gece bekletin </li><li> En az 500 ul PBS ile daha iyi her yıkayın. 3x tekrarlayın; Her zaman le de vermekinkübasyon süresi ast 5 dak. </li><li> Ikinci bir antikoru ve phalloidin çözelti hazırlayın ICC bir Alexa 488 etiketli eşek anti-fare ikincil antikoru (2 ug / ml nihai konsantrasyon) 1000 kat dilüsyonu ve Phalloidin 1000 x seyreltme (50 ng / ml nihai konsantrasyon) kullanılarak, blokaj tamponu. </li><li> PBS çıkarın ve her bir oyuğa, ikincil antikor ve phalloidin çözeltisi 300 ul ilave edin ve 2 saat boyunca inkübe ya da 4 ° C&#39;de bir gece bekletin </li><li> Yıkama başına en az 5 dakika inkübasyon ile PBS yıkama adımları tekrarlayın. Son yıkamadan olarak, 1 saat boyunca PBS içinde de her bırakın. </li><li> Bir cam slayt üzerinde anti-solmaya montaj medya tek bir damla (~ 10 ul) bırakın. </li><li> Bend sert bir yüzeye hafifçe bastırarak şırınga iğnesi, 90 ° 19 G 1 ½ ucu. Bükülmüş iğne tutun ve yavaşça iyi bir cam diski yukarı kaldırın, ve keskin forseps kullanarak diski kavramak için diğer elinizi kullanın tek bir elinizi kullanın. </li><li> Cam diski Turn &amp;# 160; cam slayt hücre yan kişiyi montaj medya icar yüz aşağı. Yavaşça cam diski yerleştirin ve oda sıcaklığında karanlık bir yerde gece boyunca kurumaya bırakın. </li><li> Görüntü immünofloresans lekeli nanopodia devam, ya da -20 ° C&#39;de slayt kutuda saklayın slaytlar Slaytlar -20 ° C&#39;de birkaç ay boyunca görüntülenebilir kalacaktır </li></ol>

<ol>
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Çıkar çatışması ilan etti.

Nanopodia sıkıca TMED aracılığıyla Matris takmak ve ortamı duygusu ve hücreler arası etkileşimleri 2,3 aracılık için polarize mobil hücreden fazla 100 mikron uzatabilirsiniz ince hücresel membran kanalları vardır. Bu yüzden sıkıca kalıntıları uzak hücre hamle olarak geride kaldığını matrix uymak Nanopodia (Şekil 1 ve 2). Nanopodia böylece bizi hücrelerin çevrelerini nasıl hissettiğinin çalışma ve hücre hareketin yolunu belirlemek için, i...

Hareket için polarizasyon esnasında, hayvan hücreleri filopodia, lamellipodia, geri çekme lifler ve fırfır 1 de dahil olmak üzere, yüzeyleri dinamik, membran uzantıların, çeşitli uzanır. Son zamanlarda bu listeye eklenir nanopodia, ince bir yeni tanınan tipi (çapı 100-300 mikron), gibi filopodia gibi F-aktin yapılarının uzatılması için membran kanalları sağlamak uzun (en fazla 50-100 mikron uzunluğunda) membran projeksiyon vardı geri çekme ve elyaf ve bu leke pozitif TM4SF1 (Transmembran-4-L-altı family-1) kültürlenmiş endotelyal ve tümör hücreleri içinde 2,3 ile. TM4SF1 ilk molekül endotel hücre proliferasyonu ve göçünde 2,3 önemli bir rol oynayan bir endotelyal hücre biyolojik olan keşfinden önce, bir tümör hücresi antijenine 4 olarak bilinen tetraspanin benzeri topolojisi ile bir proteindir. Immünofloresan boyama TM4SF1 perinuc lokalize olduğunu ortaya çıkardılear veziküller ve plazma zarına ve TM 4SF1 e nriched mikro d omains (TMED) içinde zenginleştirilmiştir. TMED çapa Matris nanopodia ve nanopodia 1-3 TMED / um uzunlukta bir düzenli aralıklı bantlı desen mevcut. Nanopodia tipik olarak bir hücrenin gelen ön uzanan ve hareket için hücre polarizasyon esnasında arka arkaya. Nedeniyle TMED bir firma yapışık doğaya, nanopodia uzak hareket olarak hücre içine geri geri edemiyoruz; terk nanopodia artıkları böylece hücresel hareketinin yolunu iz. F-aktin uzatma ve geri çekme için membran kanalları sağlamak ve hücreler arası etkileşim ve iletişimin 2,3 sitelerdir Nanopodia. Bu özellikler hücresel kutuplaşma, çevre hücresel algılama, hücre hareket yolu ve yön tayini, ve hücreler arası etkileşimler sırasında F-aktin aksamını altında yatan mekanizmaları incelemek için eşsiz bir fırsat sağlayacak nanopodia anlamına birnd iletişimi. Nedeniyle TMED oldukça hidrofobik doğa ve nanopodia ince ve kırılgan membranöz doğaya, özel bakım TMED ve nanopodia korumak için alınması gerekir. Nanopodia ve ortak laboratuvar yöntemlerle TMED çıkarılması yıkımı sadece altmış üç yayınlar 1986 1 yılında ilk keşif beri ve üzerine 100-300 mikron microdomains içinde TM4SF1 zenginleştirme bilgi eksikliği tamamlamak için TM4SF1 çıktı neden olası bir nedeni hücre yüzeyi, 2009 2, endotel hücrelerinde TM4SF1 raporu kadar. Geleneksel immüno-lekeleme yöntemleri yaygın hücreleri düzeltmek ve hücreler 5 permeabilize% 0.1 veya daha yüksek bir Triton X-100 konsantrasyonu kullanmak için, etanol, metanol veya aseton gibi organik çözücüleri kullanırlar. (I) 37 ° C% 4 PFA kullanımı ve 37 hücreleri düzeltmek: Burada anlatılan çalışmalar TMED ve nanopodia ortaya çıkarmak için geleneksel yönteme üç büyük değişiklikler uygulanmıştır76, C kuluçka makinesi, (ii) oda sıcaklığında nazik bir PBS yıkama uygulanır, ve (iii) Triton X-100, Triton X-100,% 0.03 &#39;den daha fazla özü gibi, primer antikor ilave edilmeden önce hücreler nüfuz edilebilir kılınması için sadece kısa bir süre için en az% 0.03 oranında kullanmak TM4SF1. Tüm tetraspanins hücre zarı 6 mikro bölgesini oluşturur ve bazı endotelyal ve tümör hücrelerine 2,3 in TMED ile colocalize. CD9, CD81 ve CD151 gibi tetraspanins her yerde bulunan bir ifade olarak, burada açıklanan boyama protokolü nanopodia fonksiyonunun çalışmalar için TM4SF1 olmayan pek çok farklı hücre tipleri için uzatılabilir.

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			<td><a name="RANGE!D15">0.25% Trypsin-EDTA 1X</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DMEM</td>
			<td>Life Technologies</td>
			<td>11965-092</td>
			<td>DMEM high glucose (1X), liquid, with L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">75889 5 LT</td>
			<td style="width:983px;">PBS, 10X Solution, pH 7.4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PFA (paraformaldehyde)</td>
			<td style="width:160px;">Affymetrix</td>
			<td style="width:133px;">19943 1 LT</td>
			<td>4% in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F4135-500ML</td>
			<td><a name="RANGE!D19">Fetal Bovine Serum</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">EGM2-MV</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>EGM-2 BulletKit, EBM-2 Basal Medium 500 ml and EGM-2 SingleQuot Kit Suppl. &#38; Growth Factors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">T8787</td>
			<td>Triton X-100</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">NaN3</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">S2002-25G</td>
			<td>solubilized in water. 4% stock solution and working concentration is 0.4%.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human TM4SF1 antibody&#160;</td>
			<td style="width:160px;">Millipore</td>
			<td style="width:133px;">MAB3127</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">mouse anti-human CD9 antibody&#160;</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:133px;">555370</td>
			<td>Epitope is located in extracellular domain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Alexa Fluor 488 Donkey anti-mouse 2nd antibody</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">A-21202</td>
			<td>Alexa Fluor 488 Donkey Anti-Mouse IgG (H+L)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Phalloidin</td>
			<td style="width:160px;">Chemicon</td>
			<td style="width:133px;">90324</td>
			<td>Rhodamine-conjugated Phalloidin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">anti-fade mounting media&#160;</td>
			<td style="width:160px;">Life Technologies</td>
			<td style="width:133px;">P-36931</td>
			<td>ProLong Gold Antifade Reagent with DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Buffers and solutions</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">70% ethanol&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td>17.5 ml of ethanol (200 proof) 
			7.5 ml of double deionized water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10X PBS (make 200 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20 ml of 10X PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">180 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">20% Triton X-100 (make 20 ml)&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">4 ml</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">16 ml double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">4% NaN3 (make 25 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">1 g of NaN3</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 ml of double deionized water</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">50 ng/ml bovine collagen solution in PBS (make 5 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Make stock soln. of 50 &#956;g/ml (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">830 &#956;l of Collage I (3 mg)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml PBS&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ICC Blocking Buffer (50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">49 ml PBS</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2% FBS (add 1 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td style="width:983px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">0.04% NaN3 (add 100 &#956;l of 4% NaN3)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">ICC Blocking Buffer/0.01% Triton X-100 (make 50 ml)</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">50 ml of ICC Blocking Buffer&#160;</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">25 &#956;l of 20% Triton X-100</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Cell</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">HUVEC&#160;</td>
			<td style="width:160px;">Lonza&#160;</td>
			<td>C2517A</td>
			<td><a href="http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx">http://www.lonza.com/products-services/bio-research/primary-and-stem-cells/human-cells-and-media/endothelial-cells-and-media/huvec-human-umbilical-vein-endothelial-cells.aspx</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PC3</td>
			<td style="width:160px;">ATCC</td>
			<td style="width:133px;">CRL-1435</td>
			<td><a href="http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7">http://www.atcc.org/products/all/CRL-1435.aspx#85786B46AA23451B94BC5D45200673F7</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;"></td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Equipment</td>
			<td style="width:160px;"></td>
			<td style="width:133px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">cell culture hood</td>
			<td style="width:160px;">NuAIRE</td>
			<td style="width:133px;">Nu-425-600</td>
			<td>NU-425 (Series 60) BIOLOGICAL SAFETY CABINET&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:323px;">37 &#176;C, 5% CO2 cell culture incubator&#160;</td>
			<td style="width:160px;">CellStar</td>
			<td style="width:133px;">QWJ300DABA&#160;</td>
			<td>Cellstar CO2 Water Jacketed Incubator</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">heating pad&#160;</td>
			<td>K&#38;H Manufacturing</td>
			<td style="width:133px;">1020</td>
			<td style="width:983px;">K&#38;H Lectro Kennel Heated Pad with Free Fleece Cover (http://www.amazon.com/Lectro-Kennel-Heat-Pad-22-5x28-5-In/dp/B000PSRN20/ref=pd_bxgy_petsupplies_img_y)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">centrifuge</td>
			<td style="width:160px;">Sorvall&#160;</td>
			<td style="width:133px;">T6000B&#160;</td>
			<td>Sorvall T6000 (B) Benchtop Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td style="width:133px;">Z359629</td>
			<td>Bright-Line Hemacytometer</td>
		</tr>
	</tbody>
</table>

nanopodia - thin, fragile membrane projections with roles in cell movement and intercellular interactions

Kültür yapışan hücreler hareketi ve hücreler arası etkileşimleri desteklemek için polarize durumunu korumak. Nanopodia, ince uzun, TM4SF1 (Transmembran-4-L-altı ailenin-1), imüno-lekeleme yoluyla görselleştirilebilir endotel ve kanser hücrelerinde büyük ölçüde F-aktin-negatif zar tahminleridir. 100-300 mikron çapında TMED de TM4SF1 kümeleri 14 kadar bireysel TM4SF1 moleküllere 3 ihtiva eden (TM 4SF1 e mikro d omains nriched). TMED 1 μ m &#39;başına TMED 3 ve sıkıca matrisine nanopodia çapa düzenli aralıklarla nanopodia boyunca aralıklı olarak düzenlenmiştir. Bu polarize endotel veya tümör hücrelerinin ön ön veya arka arkaya gelen daha 100 μ m uzatmak nanopodia sağlar ve MATR üzerinde geride olmak zar artıkları olurix hücre uzaklaşıyor zaman. TMED ve nanopodia nedeniyle aşırı kırılganlık ve sıcaklık hassasiyeti göz ardı edilmiştir. Rutin yıkama ve tespit yapısını bozabilir. Nanopodia lekelenme% 0.01 Triton X-100 kısa süreli maruz bırakma ve ardından, 37 ° C &#39;de paraformaldehit (PFA) doğrudan tespit ile korunur. Hücre biyolojisinde yeni ufuklar açmak Nanopodia: algılamak ve bir mesafede diğer hücreleri belirlemek, yakın temas sırasında hücreler arası etkileşimleri başlatabilir ve hareket, çoğalma dahil sinyal mekanizmaları, hücrelerin çevrelerini nasıl hissettiğinin anlayışımızı yeniden şekillendirmek ve hücre için söz veriyorum -hücre iletişimi. TM4SF1 türetilmiş nanopodia çalışmak için geliştirilen yöntemler, klasik tetraspanins, özellikle de her yerde bulunan bir ifade CD9, CD81 ve CD151 vasıtasıyla diğer hücre tiplerinde meydana nanopodia çalışmaları için yararlı olabilir.

Adım 1 için: (Örneğin HUVEC&#39;e ve bu çalışmada kullanılan PC3 olarak) hücreler normal büyümek mümkün ise, hücreler numaralı seribaşı sonra, 30 dakika içinde kollajen kaplı disk takmak Polarize ve kısa bir süre sonra mobil olmak ve ileride kendi yolunu nanopodia uzatacak hareketin. Şekil 1A ve 1B, sırasıyla bir polarize ve çoğalan HUVEC. Şekil 2A, bir cep halinde polarize PC3 hücreleri sini göstermektedir. <p ...

Watch this Scientific Journal Video about Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions at JoVE.com

Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Nanopodia bir hücrenin ön ön veya arka arka 100 mikron kadar uzanır ve hücresel ortamı anlamda ince ama kırılgan zar kanallardır. 37 ° C&#39;de, hafif bir yıkama, ve etanol, metanol veya aseton gibi bir organik çözücü önlemeyi ve daha yüksek Triton X-100 konsantrasyonları doğrudan tespiti, bu hücresel yapılar gözlemlemek için gereklidir.

Nanopodia - Cep Hareketi ve Intercellular Etkileşimleri Rolleri İnce, Kırılgan Membran Projeksiyonlar

Adherent cells in culture maintain a polarized state to support movement and intercellular interactions. Nanopodia are thin, elongated, largely ...

nanopodia-thin-fragile-membrane-projections-with-roles-cell-movement

Research

JoVE Journal

Biology

10.2K Görüntüleme.  Harvard Medical School.  Nanopodia bir hücrenin ön ön veya arka arka 100 mikron kadar uzanır ve hücresel ortamı anlamda ince ama kırılgan zar kanallardır. 37 ° C'de, hafif bir yıkama, ve etanol, metanol veya aseton gibi bir organik çözücü önlemeyi ve daha yüksek Triton X-100 konsantrasyonları doğrudan tespiti, bu hücresel yapılar gözlemlemek için gereklidir. 

Video: Nanopodia - Thin, Fragile Membrane Projections with Roles in Cell Movement and Intercellular Interactions

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Membran Biyogenez bir Lusiferaz Tabanlı Reporter Gen Testi ile incelenmesi

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Biyoloji

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

EMBL de MultiBac Protein Complex Üretim Platformu

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event that triggers the repair of mechanical wounds on the plasma membrane within ~30 sec. Recent studies revealed that mammalian cells also reseal their plasma membrane after permeabilization with pore forming toxins in a Ca2+-dependent process that involves exocytosis of the lysosomal enzyme acid sphingomyelinase followed by pore endocytosis. Here, we describe the methodology used to demonstrate that the resealing of cells permeabilized by the toxin streptolysin O is also rapid and dependent on Ca2+ influx. The assay design allows synchronization of the injury event and a precise kinetic measurement of the ability of cells to restore plasma membrane integrity by imaging and quantifying the extent by which the liphophilic dye FM1-43 reaches intracellular membranes. This live assay also allows a sensitive assessment of the ability of exogenously added soluble factors such as sphingomyelinase to inhibit FM1-43 influx, reflecting the ability of cells to repair their plasma membrane. This assay allowed us to show for the first time that sphingomyelinase acts downstream of Ca2+-dependent exocytosis, since extracellular addition of the enzyme promotes resealing of cells permeabilized in the absence of Ca2+.

Live Imaging Assay for Assessing the Roles of Ca2+ and Sphingomyelinase in the Repair of Pore-forming Toxin Wounds

Live imaging of cells exposed to the lipophilic dye FM1-43 allows precise determination of the kinetics by which pore-forming toxins are removed from the plasma membrane. This is a sensitive assay that can be used to assess requirements for Ca2+, sphingomyelinase and other factors on plasma membrane repair.

Ca Rolleri değerlendirilmesi için Görüntüleme Testi Canlı<sup&gt; 2 +</supGözenek oluşturucu Toksin yaraların tamiri&gt; ve sfingomiyelinaz

Plasma membrane injury is a frequent event, and wounds have to be rapidly repaired to ensure cellular survival. Influx of Ca2+ is a key signaling event ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Gap Junctional Hücreler arası iletişim: Doğal Ürünler Toksikantların ve Toksinler Yan Etkileri değerlendirmek için Fonksiyonel Biyomarker ve Sağlık Faydaları

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm allows for the routine imaging of nanoscale biological complexes and processes. This increase in resolution also means that methods popular in electron microscopy, such as single-particle analysis, can readily be applied to super-resolution fluorescence microscopy. By combining this analytical approach with super-resolution optical imaging, it becomes possible to take advantage of the molecule-specific labeling capacity of fluorescence microscopy to generate structural maps of molecular elements within a metastable structure. To this end, we have developed a novel algorithm &#8212; VirusMapper &#8212; packaged as an easy-to-use, high-performance, and high-throughput ImageJ plugin. This article presents an in-depth guide to this software, showcasing its ability to uncover novel structural features in biological molecular complexes. Here, we present how to assemble compatible data and provide a step-by-step protocol on how to use this algorithm to apply single-particle analysis to super-resolution images.

This manuscript uses the Fiji-based open-source software package VirusMapper to apply single-particle analysis to super-resolution microscopy images in order to generate precise models of nanoscale structure.

VirusMapper Süper çözünürlüklü Mikroskopi Açık kaynak Tek parçacık Analizi

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Flash-and-Freeze: A Novel Technique to Capture Membrane Dynamics with Electron Microscopy

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and spatial resolution on the order of ms and nm, respectively. We have developed a time-resolved electron microscopy technique, &#34;flash-and-freeze,&#34; that induces cellular events with optogenetics and visualizes the resulting membrane dynamics by freezing cells at defined time points after stimulation. To demonstrate this technique, we expressed channelrhodopsin, a light-sensitive cation channel, in mouse hippocampal neurons. A flash of light stimulates neuronal activity and induces neurotransmitter release from synaptic terminals through the fusion of synaptic vesicles. The optogenetic stimulation of neurons is coupled with high-pressure freezing to follow morphological changes during synaptic transmission. Using a commercial instrument, we captured the fusion of synaptic vesicles and the recovery of the synaptic vesicle membrane. To visualize the sequence of events, large datasets were generated and analyzed blindly, since morphological changes were followed in different cells over time. Nevertheless, flash-and-freeze allows the visualization of membrane dynamics in electron micrographs with ms temporal resolution.

We developed a novel technique in electron microscopy, &#34;flash-and-freeze,&#34; that enables the visualization of membrane dynamics with ms temporal resolution. This technique combines the optogenetic stimulation of neurons with high-pressure freezing. Here, we demonstrate the procedures and describe the protocols in detail.

Flaş-ve-Freeze: Elektron Mikroskobu ile Membran Dinamikler Yakalama Yeni Bir Tekniği

Cells constantly change their membrane architecture and protein distribution, but it is extremely difficult to visualize these events at a temporal and ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

Membran nanotüpler dev Unilamellar veziküller çekerek

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the distance and orientation of the molecules as well as the extent of overlap between the donor emission and acceptor absorption spectra. FRET permits to study the interaction of proteins in the living cell over time and in different subcellular compartments. Different intensity-based algorithms to measure FRET using microscopy have been described in the literature. Here, a protocol and an algorithm are provided to quantify FRET efficiency based on measuring both the sensitized emission of the acceptor and quenching of the donor molecule. The quantification of ratiometric FRET in the living cell not only requires the determination of the crosstalk (spectral spill-over, or bleed-through) of the fluorescent proteins but also the detection efficiency of the microscopic setup. The protocol provided here details how to assess these critical parameters.

F&#246;rster Resonance Energy Transfer (FRET) between two fluorophore molecules can be used for studying protein interactions in the living cell. Here, a protocol is provided as to how to measure FRET in live cells by detecting sensitized emission of the acceptor and quenching of the donor molecule using confocal laser scanning microscopy.

FRET'e duyarlı emisyona sahip canlı hücrelerdeki protein etkileşimlerinin değerlendirilmesi

F&#246;rster Resonance Energy Transfer (FRET) is the radiationless transfer of energy from an excited donor to an acceptor molecule and depends upon the ...

Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

This paper describes the protocols for sample preparation, data reduction, and data analysis in neutron spin echo (NSE) studies of lipid membranes. Judicious deuterium labeling of lipids enables access to different membrane dynamics on mesoscopic length and time scales, over which vital biological processes occur.

Lipid Membran Dinamiği ve Membran-Protein Etkileşimleri için Benzersiz Bir Prob Olarak Nötron Spin Eko Spektroskopisi

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane&#160;interactions, and viral ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Kardiyak ve Vasküler Düz Kas Hücrelerinin Elektrofizyolojik Çalışmalar İçin Yetişkin, Genç, Larva ve Embriyonik Zebra Balıklarından İzolasyonu

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...