The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H + Killing Seçim <ol><li> Hücre çizgileri <ol><li> Kararlı olarak NHE-eksikli hücrelerin transfekte (örneğin, CCL39 türetilmiş PS120 hücre çizgisi 16) bir fibroblast hücre çizgisinde etkili transfeksiyon verimi ve seçim üretecek memeli ifade vektörü ve transfeksiyon yöntemi herhangi bir kombinasyonu kullanılmıştır. NOT: Yıllarca Kalsiyum Fosfat yağış 19 iyi transfeksiyon verimi ile kullanılmıştır. Bu, Lipofectamine2000 gibi ticari reaktifler ile daha yakın değiştirilmiştir, transfeksiyonlar imalatçının talimatlarına uygun olarak yerine getirilmektedir. </li></ol></li><li> Çözümler <ol><li> Aşağıda tarif edilen üç steril çözeltiler hazırlayın. Filtrasyon (0.22 mikron) tarafından bu çözümleri sterilize edin. <ol><li> 50 mM NH4CI, 70 mM kolin klorid, 5 mM KCI, 1 mM MgCl2, 2 mM CaCI2, 5 mM gl içeren bir NH4 + Yükleme çözeltisi hazırlayın (pH 7.4)ucose ve pH 7.4&#39;te 15 mM MOPS. </li><li> 120 mM kolin klorür, 5 mM KCI, 1 mM MgCl2, 2 mM CaCI2, 5 mM glukoz, pH 7.0, 15 mM HEPES içeren bir durulama çözeltisi (pH 7.0) hazırlayın. </li><li> PH 7.4&#39;te 120 mM NaCI, 5 mM KCI, 1 mM MgCl2, 2 mM CaCI2, 5 mM glukoz ve 15 mM HEPES içeren bir kurtarma çözeltisi (pH 7.4) içinde hazırlayın. </li></ol></li></ol></li><li> CO2 bozabilir bu tamponlama neden olur 5% &#39;si olarak, - seçim başlamadan önce, (&quot;serbest&quot; kuluçka CO2 olarak adlandırılır) dış tanktan ek CO2 ile 37 ° C&#39; de kullanılabilen bir hücre kültürü kuluçka elde asidifikasyon. Ile yaklaşık 2 x 10 8 hücreleri (tipik olarak yaklaşık 20 konfluent 100 mm plakalar) için ilgi NHE sentezleyen hücre soyu yükseltin. </li><li> H prosedürü öldürme +: <ol><li> 3 de, yükleme Çözüm, 1 saat için ilgi hücre NHE-ifade eden hücreleri inkübeCO 2 -ücretsiz inkübatör 7 ° C. </li><li> Yükleme çözeltisi aspire ve daha sonra, yukarıda tarif edilen, durulama çözeltisi içinde iki kez yıkayın. Asitlenmesini üretecek bir dik NH 4 + gradyan oluşturulmasını zedeleyecek artık dışı NH 4 Cl ortadan kaldırmak için verimli bu adımı gerçekleştirin. </li><li> Aspirasyon ile durulama ortamı ortadan kaldırmak ve CO2 serbest inkübatör içinde 37 ° C&#39;de tekrar geri kazanım çözeltisi içinde bir saat için kuluçkalayın. </li><li> Normal kültür ortamında (% 7.5 FCS, tipik olarak DMEM) ile kurtarma ortamı yerine ve (% 5 CO2 ve% 95 hava içeren nemli bir atmosferde 37 ° C), standart kültür koşullarında hücreler büyür. </li></ol></li><li> Stabil klonlar ortaya kadar haftada iki kez seçim bu döngüyü tekrarlayın. NOT: Hücre kültürü ve seçim koşulları konusunda özel dikkat. Iş Ay o daha eğilimli herhangi bir kontaminasyon tarafından harap olabilirkültür ve tekrarlanan hücre manipülasyonlar uzun bir süre sonra CCur. <ol><li> Burada, klonlar yükseltmek ve bireysel olarak daha bölümlerde 2-3 açıklanan onları karakterize, ya karakterizasyon önce hücresel nüfus oluşturmak için onları bir araya havuz seçebilirsiniz. </li></ol></li><li> Sayaç-seçilmesi ebeveyn asit duyarlı fenotip (Şekil 1B) döndürülür olabileceği olanlar tarafından asitlenme dirençli hücreleri korumak için zaman zaman seçim (yaklaşık iki haftada) uygulayın. </li></ol> 2. Hücre içi pH Ölçümleri 2.1) Floresans pH Görüntüleme <ol><li> 490 nm&#39;de uyarıldığında, pH&#39;a duyarlı ve üretici protokollerine göre bir 445 mil izosbetik noktası sahip rasyometrik pH&#39;a duyarlı fluoresan boya BCECF / AM kullanın. <ol><li> Seçenek olarak ise, üreticinin protokollerine aşağıdaki diğer pH&#39;a duyarlı prob kullanılır. </li></ol></li><li> Bir görüntüleme set con kullanınyüksek hassasiyet video kamera bağlanmış bir ters mikroskop sisting. 450 nm ve uyarma için uygun kuvars nötr yoğunluk filtreleri ile eşleştirilmiş 490 nm dar bant girişim filtreleri ile bu seti donatın. <ol><li> Mümkünse oranı da hem λ1 ve / veya λ2 için, ya doyurarak veya çok düşük ayarlanmış bazal floresan değerlerini önlemek 1. yaklaşır, böylece λ1 ve λ2 kurulum karşılaştırılabilir floresan değerlerine filtreler ve aydınlatma koşulları uygun seti kullanın. Bu hücre içi pH değişiyor olsa oranı tespit edilmiş olmayabilir gibi o önemli eserler verebilir. NOT: Bu sistem uygun emisyon dalga boyunda (BCECF için 535 nm) hızlı perfüzyon, zaman atlamalı, yukarıda zikredilen iki dalga boylarında uyarım ve satın izin gerekir. Yazılım otomatik veri toplama ve depolama sağlayan sistemi donatın. </li></ol></li></ol> NHE İleri Faaliyet 2.2) Ölçüm (eldesiSitosolden protonlar) <ol><li> NH4 + Yükleme çözeltisi içinde 1 saat süren kuluçkalamadan hücreleri asitleştirin CO2 yokluğunda (adım 1.4). </li><li> Son beş dakika boyunca solüsyon ile BCECF-AM (5 uM nihai konsantrasyon) ilave edilir ve daha sonra hücre dışı prob ortadan kaldırmak için NH 4 + yükleme çözeltisi durulayın. </li><li> Mikroskop hücreleri monte ve istikrarlı bir temel kaydetmek için birkaç fotoğraf çekmek. Durulama çözeltisi (yukarıya bakın) serpmek. Bu asitleştirme tekabül eden floresans bir düşüşüne yol açar. </li><li> PH stabilize sonra, Li + / H +, Na + / H + veya K + / H + değişimi aracılığı ile pH yükselmesinin oranını ölçmek için aşağıdaki çözümlerden herhangi serpmek. Her bir çözelti bir potansiyel birleştirme katyon H + değişimi için test edilecek olan içerir. Bunlardan biri taşınıyorsa, bu değerden uygun olacak 495 nm&#39;de bir floresan artışı ile sonuçlanırond hücre içi pH zam için: PH 7.4&#39;de 120 mM LiCİ, 5 mM KCI, 1 mM MgCl2, 2 mM CaCl2, 5 mM glükoz ve 15 mM HEPES PH 7.4 120 mM NaCI, 5 mM KCI, 1 mM MgCl2, 2 mM CaCl2, 5 mM glükoz ve 15 mM HEPES PH 7.4&#39;de 125 mM KCI, 1 mM MgCl2, 2 mM CaCl2, 5 mM glükoz ve 15 mM HEPES </li></ol> NHE Ters Faaliyet 2.3) ölçümü NOT: Burada ilke transmembran iyonik eğimleri ters etmektir. <ol><li> Ölçümden önce, ilgi konusu hücre içi katyon ile yük hücreleri (aşağı bakınız). </li><li> BCECF / AM (bkz: adım 2.2.2) ile 5 dakika boyunca onları inkübe; , yıkayın video mikroskobu onları monte edin. </li><li> Yükleme çözüm serpmek ve istikrarlı bir temel kaydetmek için birkaç fotoğraf çekmek. </li><li> Bazal istikrar, görüntü hücreleri bir hücre dışı asidik ortam 120 m içeren perfüze olan pH değişimleri sonraE kolinklorid, 5 mM KCI, 1 mM MgCl2, 2 mM CaCl2, 5 mM glükoz ve pH değeri 6.5 olan 15 mM MES,. </li><li> LiCİ yüklemesi için, pH 7.4 ile 120 mM LiCİ, 5 mM KCI, 1 mM MgCl2, 2 mM CaCI2, 5 mM glukoz ve 15 mM HEPES oluşan bir çözelti içinde, 2 saat boyunca kuluçkalayın. Atom emme spektroskopisi ile ölçülen hücre içi lityum yaklaşık 60 mM arasında bir değer elde edilir. </li><li> Na yüklemesi için, 1mM ouabain varlığında pH 7.4&#39;te 120 mM NaCI, 5 mM KCI, 1 mM MgCl2, 2 mM CaCI2, 5 mM glukoz ve 15 mM HEPES oluşan bir çözeltiye, iki saat süreyle kuluçkalayın Na / K ATPaz engellemek için. Bu koşullar altında, hücre içi Na konsantrasyonu, 40 mM arasındadır. Not: sitosolik K + konsantrasyonu 140 mM aralığında ve bir dışarı doğru yönlendirilmiş K + gradyanı elde etmek için, bu nedenle kolay olduğu gibi K + yükleme gerekli değildir. </li></ol> 2.4) Veri Tedavi ve Kalibrasyon: Not: Bu adım ileri ve geri ölçümler için de geçerlidir. <ol><li> Her bir deneyin sonunda, 6.5 ile 7.4 arasında pH değerlerine ayarlanmış 140 mM KCI, 20 mM HEPES ve 5 uM nigerisin eden bir çözelti ile hücreleri serpmek. </li><li> Metin biçiminde altındaki görüntüleri floresan ölçümleri gibi verileri (gri düzeyleri yoğunluk seviyelerini temsil eden) ve ihracat toplayın ve bir elektronik tablo programı kullanarak aşağıda açıklanmıştır davranırım. </li><li> Her bir hücre ya da aşağıdaki denklem kullanılarak ilgili bölge için hücre içi pH değerini hesaplayın: pH = pKa + (Log (R-Rmin) / (R max R)) x F dk (λ2) / F max (λ2) </li><li> F min (λ2) / K ma x (λ2), deneyler boyunca 450 nm floresansta bir azalma ile sonuçlanır sistemi ağartıcı veya kaçak var ise, bu oran kullanın. Ancak bu, çok nadiren des kullanılan koşullarda gözlenenaçıklanan şekilde deneyleri. </li><li> Kalibrasyon verileri her pH ölçümü sonunda mevcut gibi gelen hesaplanan değerler kalibrasyon için kullanılan pH değerleri farklı olup olmadığını kontrol edin. Bu durumda, aşağıdaki yordamı kullanarak kalibrasyon tüm deneysel belirlenen pH değerleri ayarlayın: <ol><li> Aşağıdaki katsayısı (Q, kalibrasyon değerleri arasında ölçülen değerler / Fark arasındaki fark) hesaplayın. Hesaplanan değerler karşılık gelen kalibrasyon değerlerini eşit olacak şekilde Q ile bütün veri kümesi ilave veya çıkarma ile son ayarlamayı yapın çarpın. </li></ol></li></ol> Hızlı Li + Alımına tarafından NHE7 başlangıç ​​Oranları 3. Ölçümler <ol><li> Çok yuvalı plakalar üzerinde Tohum hücreleri (6 yanı sıra 24) üzerinde ya da 20 de tarif edilen alternatif olarak nigericin / sığır serum albümini (BSA) asitlendirme tarif NH4 + yükleme tekniği kullanarak bunları asitleştirmek. </li><li&gt; Durulama Plakalar iki kez hızla yukarıda tarif edilen durulama çözeltisi kullanılarak ve daha sonra arzu edilen miktarda (hafif, Km değerlerinde altında) 1-10 mM lityum içeren alım Çözeltilerin hücreleri ve katyon istenen konsantrasyonlar ve / veya ilgi duyulan inhibitörleri inkübe zaman. </li><li> Bu taşıma başlangıç ​​oranları koşullarda faaliyet sağlamak için kısa ve tutarlı alım süreleri (genellikle bir dakika veya altında) koruyun. Ayrıca alımı için kullanılan tüm çözümler izotonik olduğundan emin olun. </li><li> Alım süresi sonunda, dikkatli bir şekilde alımı ortamı ortadan kaldırmak ve buz soğukluğunda fosfat tamponlu tuzlu su (PBS) ile hücreler dört kez yıkayın. (Dördü ideal için az 10 sn) lityum akışını önlemek için mümkün olduğunca hızlı bu yıkar gerçekleştirin. </li><li> Lyse% 25 Nitrik asit hücreler. % 25 nitrik asit, oyuk başına 250 ul uygulanır ve daha sonra pipet ucu ile her bir oyuğa hurda ve yeni mil bütün iyi süspansiyonu transfer, en az 1 saat bekletincrocentrifuge tüp. </li><li> Selüler artığın ayrılması için 15,000 x g&#39;de (oda sıcaklığı), 5 dakika boyunca, onları santrifüj, bir 1.5 ml mikrosantrifüj tüplerine lizat aktarın. </li><li> Atomik absorpsiyon spektroskopisi 21 ile süpernatanların lityum içeriğini ölçün. NOT: Farklı şirketler Lityum oyuk katot lambası ile donatılmış gereken atomik absorpsiyon spektrometre sağlamak. Bu makineler çok hassas ama aynı zamanda oldukça hassas olarak üreticinin talimatlarını izleyin. </li></ol>

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The authors have nothing to disclose.

Bu protokol, mevkiye-yönelik mutagenez ile birincil dizisini değiştirmeden, plazma membranında, hücre içi, Na + / H + değiştiriciler ifade eden hücreleri seçin açıklamaktadır. Bu değiştirici hemen karakterize edilebilir. Bu yöntem, plazma membranında veziküler, Na + / H + değiştiricileri ifade edecek hücre hatları seçmek için hücre içi proton hücresel zehirlilik dayanmaktadır. Bu örneğin, Li +, K +,</su...

Çoğu hücre içi bölmeleri olgunlaşması, insan ticareti, proteinler veya hormonlar ve yükleme nörotransmitterlerin geri dönüşümü için önemli bir parametredir asidik lümen pH görüntüler. Bu sitosol ve vesiküler içeriği arasındaki pH gradyanı veziküler CIC klorür taşıyıcılar 2 bağlanmış, vaküoler H + ATPazların 1 ile elde edildiği gösterilmiştir. Her iki knock-out (KO) farelerde ve insan hastalarda, bu taşıyıcıları önemi onların genlerinde 3-6 mutasyonlar neden ağır fenotip tarafından vurgulanmıştır. Sodyum-hidrojen değiştirici SLC9A ailesinin üyeleri, aynı zamanda, Na + / H + değiştirici NHEs olarak adlandırılan hücre-içi pH ve hücre hacmi düzenlenmesinde anahtar etkileyiciler yanı sıra epitelden asit-baz eşdeğerleri vektör taşıma olduğu gösterilmiştir . Plazma zarı NHEs yanı sıra, üç yüksek + / H + değiştiriciler, NHE-6, 7, Na korunmuşve 9, trans-Golgi ağı ve erken endozomlarda 7 olarak ifade edilmiştir. Onların genlerinde mutasyonlar Angelman benzeri veya Christianson sendromları 8-9, aile temelli otizm 10 ve Dikkat Eksikliği Hiperaktivite Bozukluğu 11-12 ile bağlantılı olmuştur. Bu dönüştürücüler ayrıca Alzheimer hastalığı yatkınlık 13 ve X-bağlantılı mental retardasyon bitişik genler gibi nörodejeneratif sorunlara 14 sendromları dahil edilmiştir. Birlikte ele alındığında, bu çalışmalar beyin gelişimi ve / veya fonksiyon bu hücre içi NHEs önemini vurgulamak. Bu eşanjörleri hücre içi lokalizasyonu doğru onların iyon seçicilik ölçümleri, ulaşım yönü, kinetik parametreleri, ve düzenleme önler. Hücre içi bölümlerinde ifade tüm nakliyeciler için olduğu gibi, onların biyokimyasal faaliyetlerini değerlendirmek ve dolayısıyla tam olarak fizyolojik rolleri ve Mechan anlamak son derece zorizm kendi patolojik etkileri altında yatan. Yüksek sitozolik K + konsantrasyonu dayanarak, en çok yaygın kabul gören hipotez onlar K + birleştiğinde proton akış taşıyıcıları olarak görev olmasıydı. Bu değer sabit bir veziküler pH&#39;sını muhafaza etmek için V-ATPazların proton pompalama telafi edilebilir bir proton sızıntı olması, hipotez edilmiştir. Bu görsel maddenin amacı, plazma membranında veziküler taşıyıcıları ifade, ve (ii) bu nakil fonksiyonlarını ölçmek için, bağımsız iki yaklaşım göstermek için hücre çizgilerinin genetik olarak seçilmesi ile sağlayan bir yöntemi göstermek için, (i) &#39;dir. Üç yıl önce, Pouysségur ve Franchi NHE ailesinin 15 üyesi moleküler klonlama ve karakterizasyonu etkin bir genetik yaklaşım öncülük etmiştir. Bu tarama yöntemi olarak hücre içi proton toksisite dayanıyordu. İlk adım hücre hatları eksik elde etmek olduHerhangi bir Na + / H + değişimi bu taşıyıcı geri döndürülme oranı kullanılarak, plazma membranında ifade edilmiştir. Fibroblastlar (CCL39 hücre çizgisi), Na + ya da Li + önceden yüklenmiş ve daha sonra 2 saat süre ile bir asidik hücre dışı ortam (pH 6.5) içine yerleştirildi. Bu fonksiyonel, Na + / H + değişimi ifade eden hücrelerin ölümüne ve damar sertliği-yetersizliğine sahip hücrelerin (PS120 hücre çizgisi) 16 seçimine yol açmıştır. Bikarbonat içermeyen bir ortam içinde kültive edilince, bu hücreleri, akut hücre içi asitlenme çok hassastır. Bu tür hücreleri, akut hücre içi acidifications sunulur Sonuç olarak, eğer plazma membranında herhangi bir işlevsel proton akış mekanizması sentezleme pozitif (bakınız 17) seçilir. Bu gibi asitleştirme teknikler plazma membranında WT hücre içi NHEs zorla ifade sağlayan kusurları trafiğe sahip hücre çizgileri izole etmek için kullanılabilir. Ökaryotik, Na + / H deEşanjörleri elektro +, onlar kanalları ölçmek için büyük bir başarı ile kullanılmaktadır elektrofizyolojik yaklaşımlarla ölçülebilir değildir. Bu yazıda, bu nedenle hücre içi pH ölçümleri ve lityum alımının hızlı kinetik bu değiştirici aktivitesini ölçmek için nasıl gösterir. Temel kavramlar aynı gibi, bu seçim bölümü için geliştirilmiş süreçlerin birçok fonksiyonel ölçümler için doğrudan kullanılan olduğunu fark etmek ilginçtir. İlginç bir şekilde, bu metinde tarif edilen yaklaşımı kullanılarak seçilmiş hücre çizgileri mevcut trafik kusur veziküler potasyum kanalı TWIK1 18 olarak plazma zarının diğer veziküler proteinlerin daha büyük bir ifadesine neden olduğu gözlenmiştir. Bu veziküler transmembran proteinler için genel bir tutma kusur mekanizmasının seçimi doğru işaret. Dolayısıyla bu seçim prosedürü ve Mayıs üretir hücrelerihücre içi bölmelerin zar proteinleri üzerinde çalışanlar için ümit veren bir araç oluşturmaktadır. Yanı sıra burada sunulan ölçüm teknikleri olmayan diğer Elektrojenik taşıyıcıları çalışmak için geçerli olabilir.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard cell culture equipement</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td>Used for H+ killing selection composed of many devices with different catalog numbers</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator with CO2</td>
			<td style="width:119px;">Sanyo</td>
			<td style="width:119px;">MCO15A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Incubator without CO2</td>
			<td style="width:119px;">Heraeus instrument</td>
			<td style="width:119px;">BB6220</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laminar flow hood PSM1200NF</td>
			<td style="width:119px;">Fisher&#160;</td>
			<td style="width:119px;">52010120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM medium</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">D5796</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS gold</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:119px;">A15-151</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicilin/streptomycin</td>
			<td style="width:119px;">PAA</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 10X</td>
			<td style="width:119px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
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			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Atomic Absorption spectrometer with Zeeman furnace system</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:119px;">ICE 3500 GFZ</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">LiCl</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">L4408</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitric Acid</td>
			<td style="width:119px;">sigma</td>
			<td align="right" style="width:119px;">438073</td>
			<td></td>
		</tr>
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			<td></td>
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			<td height="42" style="height:42px;width:216px;">Fluorescence videomicroscopy set</td>
			<td style="width:119px;">Leica</td>
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			<td>Composed of many devices with different catalog numbers</td>
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			<td style="width:119px;">DMI6000B</td>
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			<td height="21" style="height:21px;width:216px;">incident fluorescence</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888901</td>
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			<td height="21" style="height:21px;width:216px;">motor bracket</td>
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			<td align="right" style="width:119px;">11888379</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LED transmission light&#160;</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">8097321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">motorized plate</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11522068</td>
			<td></td>
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			<td></td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">camera output</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888373</td>
			<td></td>
		</tr>
		<tr height="21">
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			<td align="right" style="width:119px;">11507807</td>
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			<td height="42" style="height:42px;width:216px;">images acquisition/analysis software</td>
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			<td align="right" style="width:119px;">11888375</td>
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			<td height="21" style="height:21px;width:216px;">optics</td>
			<td style="width:119px;">leica</td>
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			<td height="21" style="height:21px;width:216px;">fluorescence Xenon lamp</td>
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			<td height="21" style="height:21px;width:216px;">camera</td>
			<td style="width:119px;">hamamatsu</td>
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			<td height="21" style="height:21px;width:216px;">metafluor/Mmfluor software</td>
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			<td align="right" style="width:119px;">11640905</td>
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			<td height="21" style="height:21px;width:216px;">pH sensitive probe, BCECF-AM</td>
			<td style="width:119px;">life technologies</td>
			<td style="width:119px;">B1170</td>
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			<td height="21" style="height:21px;width:216px;">Nigericin</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">N7143</td>
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functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

Endozomal asidifikasyonu gibi sinaptik vesiküller protein geri dönüşüm ve bozulma, reseptör duyarsızlaştırma ve nöro-yükleme gibi işlemler, geniş bir aralığı için önemlidir. Bu asitlenme CIC klorür nakliyecilere birleştiğinde proton ATPazların, aracılık tarif edilmektedir. Elektro-proton taşıyıcıları yüksek ölçüde muhafaza edilmiş, Na + / H + değiştiriciler (NHE) 6, 7 ve 9, bu bölmelerin olarak ifade edilmiştir. Genlerindeki mutasyonlar, insan bilişsel ve nörodejeneratif hastalıklar ile bağlantılı olmuştur. Onların hücre içi lokalizasyonu detaylı fonksiyonel karakterizasyonu engelledi gibi Paradoksal, rolleri, zor kalır. Bu yazının bu sorunu çözmek için bir yöntemi gösterir. Bu plazma membranında hücre içi NHEs tutma akut sitosolik asitlenme kalan kapasitesine sahip mutant hücre hatlarının seçimi oluşur. Daha sonra iyon seçiciliği ve aktivitesini ölçmek için iki tamamlayıcı protokol göstermektedir(i) bir floresan görüntü mikroskopi kullanılarak hücre içi pH ölçümleri göre, ve (ii) bir lityum alımının hızlı kinetik göre: Bu değiştirici. Bu tür protokoller olmayan diğer Elektrojenik taşıyıcıları ölçmek için çıkarım olabilir. Ayrıca, burada sunulan seçim prosedürü bir hücre içi tutma arızalı fenotip hücreleri üretir. Bu nedenle, bu hücreler de plazma zarı diğer veziküler membran proteinleri ifade edecektir. Burada tasvir edilen deneysel strateji, bu nedenle daha sonra veziküler + / H + seçimi için kullanılan değiştiriciler, Na ile birlikte, plazma membranında ifade edilecektir diğer hücre içi proteinleri incelemek için potansiyel olarak güçlü bir araç olarak kullanılabilmektedir.

Seçim: Şekil 1A&#39;da görüldüğü gibi, lH + öldürme seçimi, amonyum zayıf bir baz difüzyon dayanmaktadır. zayıf bazlar ve hücre içi pH üzerindeki asitler difüzyon etkisi Walter Bor ve işbirlikçileri 22 öncülük edilmiştir. Zarif bir fikir pozitif genetik seçimi için ölümcül bir asitlenme sonra Jacques Pouysségur 17 tarafından geliştirilen üretmek için bu olguyu kullanmak için. Böyle bir protokol kapsamında,...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Na fonksiyonel karakterizasyonu<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Seçim Proton-öldürme Kullanma hücre içi Bölmeler ve Eşanjörleri Plazma Membran Them Express

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

functional-characterization-na-h-exchangers-intracellular

Research

JoVE Journal

Biology

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

9.2K Görüntüleme.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

Video: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

Astrositler Plazma Membran ve Küresel Hücre içi kalsiyum Dynamics civarında Ölçüm

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Biyoloji

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidik Mesophases kullanarak Yapı Tayini için Membran Proteinleri Kristalize

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Akış Sitometri Plazma Membran GLUT4 Translokasyon Kantitatif Ölçüm

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

Endojen Wild-tip BRCA1 Express hücreler kullanarak Homolog Rekombinasyon BRCA1 Mutasyonlar Etkileri belirlenmesi

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Lipidik Mesophases Membran Proteinleri Yüksek throughput Kristalizasyon için bir robot kullanımı

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Makromoleküler Kristalografi tarafından Yapı Tayini için lipidik Mesophases Yetiştirilen Membran Proteinlerinin Hasat ve Cryo-soğutma Kristaller

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Floresan Proteinler ifade Canlı Farelerde Görüntüleme hücrealtı Yapıların Intravital Mikroskopi

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

PTIRFM ile görüntüleme Plazma Membran deformasyonlar

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Zararlı etkileri, düşük basınç plazma sterilizasyon Bacillus subtilis sporlar kullanarak canlı cep mikroskobu sağkalıma soruşturma

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...