The authors would like to thank Dr. Anas Kamleh for valuable help with optimizing mass spectrometry methods, and Annika von Vollenhoven for assistance with cell sorting. This research was supported by the Swedish Foundation for Strategic Research (grant no. FFL12-0220) and the Strategic Programme in Cancer Research (IR, RN); the Swedish Heart-Lung Foundation (CEW, HG); and Mary Kay Foundation (JW, MJ).

1. Metabolit Ekstraksiyon <ol><li> Çanak Ekstraksiyon <ol><li> izotop etiketli kültür ortamı + diyaliz takviyeleri (serum veya diğer büyüme takviyeleri) içinde üç kopya halinde bir 6-yuvalı plaka içindeki kültür hücreleri hücreler% 75 konfluent hale gelene kadar. Not: U- 13 C-Glukoz% 40 ihtiva eden RPMI içinde 48 saat ve% 70 U 13 C, 15, N2-glutamin ve% 5 diyaliz edilmiş FBS (Fetal Sığır Serumu) için Buraya Kültür HeLa hücreleri. Diyaliz FBS etiketlenmiş ortamı kontamine olabilir küçük molekül ağırlıklı metabolitleri kurtulmak için kullanılır. gerçek deneyden önce diyaliz ek hücrelerin kültürlenmesi Hücreler ortam içinde normal olarak büyüme sağlamak için önerilir. Takviyeler, gece boyunca yılan derisinden diyaliz tüpü kullanılarak 0.15 M NaCI çözeltisi içinde diyalize edilmiştir. </li><li> Ekstraksiyon ıskarta kültür medya gününde, 500 uL soğuk HBSS ile iki kez kuyu durulayın ve sonra atın. NOT: Burada HBSS% 40 içeren kullanın U-13 C-Glukoz bu kültür ortamında da olduğu. </li><li> Ekle 600 uL% 100 metanol, kuru buz üzerinde ön soğutulmuştur. </li><li> kuru buz ve bir hücre kazıyıcı ile hücre malzemeyi çıkarmak için çanak aktarın. </li><li> Dikkatle kütle spektrometresi analize kadar -80 ° C &#39;de, bir mikrosantrifüj tüpü ve saklamak için hücre ekstreleri pipetle. </li></ol></li><li> Sahte sıralanmış hücrelerinin alınması <ol><li> izotop etiketli kültür ortamı + diyaliz takviyeleri üç kez 100 mm tabak kültür hücreleri. Not: 100 mm tabak 48 saat süreyle burada kültür, HeLa hücreleri, hücre sayısının (~ 4 x 10 6) 500,000 kriteri hücreleri elde etmek için gereklidir, çünkü. HeLa hücreleri özü Bu sayı metabolitlerinin iyi bir ölçüm elde etmek için gerekli oldu. Kullanılan kültür ortamı RPMI% 40 U 13 C-Glikoz ve% 70 U 13 C, 15, N2-glutamin ve diyaliz FBS,% 5 idi. </li><li> ekstraksiyon, ıskarta kültür ortamı gününde, w kuyu durulayıni 1.5 ml ılık HBSS ve sonra atın. </li><li> 37 ° C&#39;de 4 dakika için 1.5 ml tripsin / EDTA ekleyerek hücreleri ayırın. 4 ° C veya buz aşağıdaki adımları uygulayın. </li><li> 3 mL buz gibi soğuk HBSS (Hank Dengeli Tuz Çözeltisi) + diyaliz ek eklenerek tripsin devre dışı bırakır. </li><li> 3 dakika boyunca 750 x g&#39;de 15 ml tüp ve santrifüj hücreleri toplamak. </li><li> 1-2 x 10 6 hücre / mL, tek hücre elde etmek için 40 mikron hücre süzgeçler geçmesine ve 5 ml tüp transfer konsantrasyonunda HBSS + diyaliz ek + 1 mM EDTA içinde pelletini. </li><li> Sıralama, sadece tekli grubu için bu hücre sıralayıcı gating arasındaki hücreler, 4 ° C&#39;de 3 dakika boyunca 750 x g&#39;de santrifüj kriteri hücreleri. NOT: Burada sıralama HeLa enstrüman basıncı 27 psi ile bir oran 1000 olayları / s hücreleri, ve 100 mikron memesi kullanarak. HeLa hücreleri büyük yüzden mümkün olduğunca sağlam pelet almak için yavaş bir olay hızı ve geniş memede tasnif edilmelidir. throughou soğuk blok hücreleri tutunt metabolizmayı azaltmak için sıralama. Sıralama prosedürün ayrıntılı bir açıklama, daha önce 12 tarif edilmiştir. </li><li> Süpernatantı atın ve metanol eklemeden önce homojen bir pelet elde etmek için 50 uL buz dH 2 O pelletini. şirketinden pelet soğuk metanol eklenmesi tekrar süspansiyon zor katı pelet oluşturur. </li><li> kuru buzda muhafaza 540 uL metanol ilave edilerek metabolitleri ekstrakte ve sıvı kromatografisi kadar -80 ° C özler devam - yüksek çözünürlüklü kütle spektrometrisi (LC-HRMS) analizi. </li></ol></li><li> Hücre döngüsünün Ekstraksiyon hücrelerini kriteri <ol><li> Kültür ortamı + diyaliz takviyeleri üç kez 100 mm tabak kültür hücreleri. NOT: Geminin Fucci Yeşil G1 (negatif) ve SG2M (pozitif) hücrelerin sıralama için izin verir (MAG1-hGem) sondası 13 içeren Burada kültür HeLa hücreleri. Hücreler için izotop izleme ortam içinde kültive edilebilirgerektiği gibi uzun s. Burada etiketsiz medyada 46 saat boyunca onları kültürlü var, o zaman biz RPMI% 40 U- 13 C-Glikoz ve% 70 U 13 C içeren açtınız, 15 N2-Glutamin 2 saat önce sıralama başlangıç. Bu kısa bir darbe etiketleme gerektiren hücre döngüsü evreleri hakkında çalışma yapabilmek amacıyla yapıldı. </li><li> ekstraksiyon, ıskarta kültür ortamı gününde, 1.5 mL sıcak HBSS ile kuyu durulayın ve sonra atın. </li><li> 37 ° C&#39;de 4 dakika için 1.5 ml tripsin / EDTA ekleyerek hücreleri ayırın. 4 ° C veya buz aşağıdaki adımları uygulayın. </li><li> 3 ml buz HBSS + diyaliz takviyesi ekleyerek tripsin devre dışı bırakın. </li><li> 3 dakika boyunca 750 x g&#39;de 15 ml tüp ve santrifüj hücreleri toplamak. </li><li> 1-2 x 10 6 hücre / mL, tek hücre elde etmek için 40 mikron hücre süzgeçler geçmesine ve 5 ml tüp transfer konsantrasyonunda HBSS + diyaliz ek + 1 mM EDTA içinde pelletini. </li><li> Hücre sıralayıcı g yoluyla sırala hücreleriDaha sonra, enkaz ve çiftleri dışarı ating ilgi hücre markeri için yolluk ve santrifüj sıralanmış hücreler 4 ° C&#39;de 3 dakika boyunca 750 xg&#39;de. NOT: Burada sıralama HeLa enstrüman basıncı 27 psi ile bir oran 1000 olayları / s hücreleri, ve 100 mikron memesi kullanarak. metabolizma hızını azaltmak için sıralama boyunca soğuk bloklar hücreleri tutun. </li><li> Süpernatantı atın ve metanol eklemeden önce homojen bir pelet elde etmek için 50 uL buz dH 2 O pelletini. şirketinden pelet soğuk metanol eklenmesi tekrar süspansiyon zor katı pelet oluşturur. </li><li> kuru buzda muhafaza 540 uL metanol ilave edilerek metabolitleri ekstrakte ve LC-HRMS analize kadar -80 ° C &#39;de özler tutun. </li></ol></li></ol> 2. Kütle Spektrometre Analiz Not: Burada bir LC-HRMS sistemde hücre özleri analiz etmek için protokol açıklar. Hücre ekstrelerinin analizi için herhangi bir metabolomiks yöntemleri kullanılabilir. Tam taramaAnaliz metabolitlerin geniş bir tespit edilmesi için yararlı olabilir. <ol><li> Bir kütle spektrometresi referans kalibrasyon karışımı kullanılarak aleti kalibre edin. </li><li> 15 saniye için 30 dakika ve vorteks için buz üzerinde çözülme hücre ekstreleri. </li><li> 4 ° C&#39;de 13,000 x g&#39;de 10 dakika süre ile bir eğirme filtresi ve santrifüj hücre ekstresi 100 uL aktarın. </li><li> LC-HRMS sistemi üzerine süzüntü 12.5 mcL enjekte edilir. </li><li> (ZIC®-HILIC) kolonu (150 mm x 4.6 mm, 5 um partikül ebatlı) bir zitteriyonik Hidrofilik interaksiyon sıvı kromatografisi kullanılarak ayrı metabolitler% 0.1 formik bir gradyan elüsyonu kullanılarak ZIC®-HILIC kılavuz sütunun (20 mm ile 2.1 x) ile donatılmış su içinde asit (solvent A) ve asetonitril (çözücü B). Çözücü A% 20 gradyan elüsyonu başlatın ve 17 dakika içinde% 80 kadar arttırır. Sırasıyla, en az 400 uL akışı 1 ve kolon sıcaklığı ve numune tablası 23 ° C&#39;de ve 4 ° C&#39;de 4 dakika boyunca bu oran muhafaza edin. </li><li> Bir i kullanınmetabolitler algılama, iyonizasyon kaynağı olarak pozitif ve negatif mod hem de ısıtılmış bir elektrosprey (lH-ESI II) ve 70.000 tam genişliği yarı maksimum (FWHM) içindeki bir kütlenin güçte tam tarama tedarik etme kipinde kromatografik ayırma bağlanmış Nstrument ( m / z 200). </li><li> akış 45 oranında, 350 ° C&#39;de 10 Au (rasgele birim) ve set buharlaştırıcı sıcaklığında ve pozitif mod ve -3.5 kV 4 kV elektrosprey voltajında ​​kılıf gaz ve yardımcı gaz için azot (saflık&gt;% 99.995) kullanarak negatif modda. </li></ol> 3. Veri Analizi <ol><li> standartlar mevcuttur ve numunelerde kaliteli zirveleri göstermek hangi metabolitler bir sayı seçin. Kaliteli zirveleri gürültü oranı yüksek sinyal var. Zirve kalite kontrol ve yanlış izotopları dahil değil emin olmak çok önemlidir. şekil ve / veya saklama süresi farklı izotopik tepe muhtemelen yanlış. </li><li> izotop etiketli örnekleri (kütle isotopomer hesaplamak içinMI) Tüm MIS toplam pik alanları ile her MI pik alanı bölerek kesirler. </li><li> Olarak, sırasıyla, 13 ° C ve 15 N, zenginleştirme etiketli karbon / azot fraksiyonları hesaplayın <img alt="denklem 1" src="/files/ftp_upload/55011/55011eq1.jpg" /> burada, n, metaboliti içindeki toplam karbon sayısı (ya da nitrojen, sırasıyla) &#39;dir, ve karışımı X MI bölümüdür. NOT: Tüm hesaplamalar programlama dilleri kullanılarak yapılabilir. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Bizim yöntemi hücresel metabolitleri MID bir hücrenin metabolik faaliyetleri &quot;tarihini&quot; yansıtması ilkesine dayanmaktadır. Bu, daha önceki bir hücre ayırma prosedürü, hücrelerin karmaşık eden oluştu şekilde mümkün hücrelerin alt popülasyonunda metabolik faaliyetlerini araştırmak için yapar. Buna karşılık, metabolitleri pik alanları sıralanmış hücreler ve kültür çanak 11 doğrudan çıkarma özleri arasında belirgin farklılıklar göstermektedir. Böl?...

Yüksek organizmalar daha karmaşık işlevleri hakkında getirmek için işbirliği farklı hücre tiplerinin karmaşık topluluklar içerir. Örneğin, tümörler de sadece kanserli hücreleri, ancak fibroblastlar, kan damarlarını oluşturan ve genellikle bağışıklık hücresi 1 infiltratlarının hücreleri içerir; Kan bağışıklık hücresi alt tiplerinin 2 onlarca karmaşık bir karışımını içerir; ve hatta kültürlenmiş hücre çizgileri, luminal ve göğüs kanseri hücrelerinin 3 taban alt tipleri gibi birden fazla alt popülasyonlarının, oluşabilirler. Metabolik &quot;işbirliği&quot; sergileyebilir arada Dahası, farklı hücre tipleri. Örneğin, beyinde, astrositler bu alt tabakayı 4 okside nöronlara ardından &quot;beslenen&quot; dir laktat, glikoz dönüştürmek için düşünülen; T lenfositleri sistein 5 kaynağı olarak bitişik dendritik hücreler göre bazı bağlamlarda vardır; ve kanser hücrelerinin Asso ile işbirliği yapabilirtümör 6 kaygılar da fibroblastlar. Bu tür sistemlerin metabolik davranışını anlamak için, ayrı ve mevcut olan çeşitli hücre tipleri metabolik etkinliklerini ölçmek için gereklidir. Bugüne kadar hücre türlerini ayırmak için en yaygın kullanılan yöntem floresan aktive hücre sıralama olduğunu. Bu yöntem, hücre tipi veya ilgi durumu floresan antikorlar, işlenmiş floresan proteinleri ifade ya da diğer boyalar kullanarak &quot;etiketli&quot; koşuluyla, geniş çapta uygulanabilir. Bir seçenek hücre sıralayıcı ile birbirinden bağımsız olarak hücre türleri, yeniden kültür elde edilen ayrı hücre tipleri için, ve daha sonra bu kültürler 7 metabolizması çalışmalar yapmak. Hücre tipi veya fenotip kültür şartlarında stabildir ve hücre döngüsü devletler, ne de ko-kültürlerde metabolik işbirliği gibi geçici davranış yakalama olamaz Ancak, bu sadece uygulanabilir. Bu gibi durumlarda, metabolizma, böylece doğrudan ölçülmelidirrted hücreleri. Bu prosedür sıralama hücre kendi metabolizmasını 8 bozulabilir gerilimlere hücreleri tabi çünkü zorlu ve biz bu yaklaşımı 9, 10 alarak sadece bir kaç çalışmalar farkındayız. Özellikle, mutlak metaboliti bolluğu ölçümleri artık güvenilir böylece amino asitler gibi önemli metabolitleri, hücre sıralama tamponunda tutulan hücrelerden sızabilir bulduk 11 (sıralı fraksiyonları arasındaki göreceli karşılaştırma hala değerli olabilir rağmen). Bu sorunları aşmak için, biz sıralama öncesinde duraylı izotop hücreleri etiket ve hücresel metabolitleri yerine metabolit bolluklarının MID odaklanmak. MID uzun zaman ölçeklerinde üzerinde oluştuğu için, daha az sıralama koşullara kısa süreli maruz kalma etkilenen edilmelidir. Biz DA sağlayacak kadar hassastır tam tarama yüksek çözünürlüklü kütle spektrometresi kullanılarak MID ölçmekhücre sıralama zamanın yaklaşık 30-60 dk gerektiren yaklaşık 500.000 sıralanmış hücrelerden başlayarak metabolitlerin yüzlerce, üzerinde ta. kültür çanak gözlenen MID orijinal kültür bulunanların temsil olmasını sağlamak için yapılır bir arasında bir karşılaştırma doğrudan kontrolü (herhangi bir özel nüfus yolluk olmadan cep sıralayıcı enstrüman geçirilerek hücreleri) ve metabolit çıkarma &quot;mock sınıflandırılmaktadır&quot;. kararlı izotop izleyicilerin seçimi bağlı olarak, çeşitli metabolik yollar bu yöntemle ele alınabilir.

<table border="0" cellpadding="0" cellspacing="0" width="1001">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:420px;">HBSS</td>
			<td style="width:165px;">Sigma</td>
			<td style="width:139px;">H6648</td>
			<td style="width:279px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">INFLUX (inFlux v7 Sorter)</td>
			<td style="width:165px;">BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-13C-Glucose</td>
			<td>Cambridge isotopes</td>
			<td>40762-22-9 / GLC-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">U-13C,15N2-Glutamine</td>
			<td>Cambridge isotopes</td>
			<td>CNLM-1275-H-0.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol (JT Baker), HPLC grade</td>
			<td style="width:165px;">VWR</td>
			<td style="width:139px;">BAKR8402.2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrafree - MC - VV centrifugal Filters. Durapore PVDF 0.1 &#181;m</td>
			<td style="width:165px;">Millipore</td>
			<td style="width:139px;">UFC30VV00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultimate 3,000 UHPLC</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Q-Exactive Orbitrap Mass spectrometer</td>
			<td style="width:165px;">Thermo Fisher scientific</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Merk-Sequant ZIC HILIC column (150 mm x 4.6 mm, 5 &#181;m)</td>
			<td style="width:165px;">Merck KGaA</td>
			<td style="width:139px;">1.50444.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Merk-Sequant ZIC HILIC guard column (20x2.1 mm)</td>
			<td style="width:165px;">Merck KGaA</td>
			<td style="width:139px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile Optima LC-MS, amber glass</td>
			<td style="width:165px;">Fisher Scientific</td>
			<td style="width:139px;">A955-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Milli-Q water</td>
			<td style="width:165px;">Millipore</td>
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			<td>Produced with a Milli-Q Gradient system</td>
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			<td style="width:165px;">Sigma-Aldrich</td>
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			<td>Calibration kit</td>
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			<td height="21" style="height:21px;">SNAKESKIN 10K MWCO&#160;</td>
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			<td height="21" style="height:21px;">Mathematica v.10&#160;</td>
			<td style="width:165px;">Wolfram Research</td>
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a method for measuring metabolism in sorted subpopulations of complex cell communities using stable isotope tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

Bir örnek olarak, burada, hücre döngüsü faz göre düzenlenmiş HeLa hücrelerinin metabolizmasını araştıran bir deney tarif eder. Her iki karbon ve nitrojen merkez metabolitlerin geniş bir etiket için, U-13C-glükoz ve U, 13C, izleyiciler olarak 15 N-glutamin ile 48 saat boyunca hücrelerin kültürü yapıldı. Izotopların ara seviyeleri daha değişik MI desen 14 üretmek eğilimindedir doğrulama deney için zengin ...

Watch this Scientific Journal Video about A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing at JoVE.com

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Kararlı İzotop izlemeyi kullanma Kompleks Hücre Toplulukları Sıralama alt popülasyonlar Metabolizma Ölçme Bir Yöntem

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

a-method-for-measuring-metabolism-sorted-subpopulations-complex-cell

Research

JoVE Journal

Biochemistry

8.2K Görüntüleme.  Karolinska Institutet. This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

Video: A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

A modified northern blotting method for measuring N6-methyladenosine (m6A) modifications in RNA is described. The current method can detect modifications in diverse RNAs and controls under various experimental designs.

RNA ölçülmesi için bir yöntem<em&gt; K</em<sup&gt; 6</supHücre ve dokularda&gt; -methyladenosine Değişiklikler

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

Biyokimya

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

bir su-içinde-yağ emülsiyonu santrifüj Dev veziküller Kapsüllenen Mikrokürelerin hazırlanması

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Hydrolysis of a Ni-Schiff-Base Complex Using Conditions Suitable for Retention of Acid-labile Protecting Groups

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide sequences. Synthesis of some unnatural amino acids often includes the use of a precursor consisting of a Schiff-base stabilized by a nickel cation. Unnatural side-chains can be installed on an amino acid backbone found in this Schiff-base complex. The resulting unnatural amino acid can then be isolated from this complex using hydrolysis of the Schiff-base, typically by employing reflux in strongly acidic solution. These highly acidic conditions may remove acid-labile side-chain protecting groups necessary for the unnatural amino acids to be used in microwave-assisted solid-phase peptide synthesis. In this work, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff base complex. Hydrolysis conditions presented in this work are suitable for retention of acid-labile side-chain protecting groups and may be adaptable to a variety of unnatural amino acid substrates.

Here, we present an efficient hydrolysis and subsequent Fmoc protection of an amino acid isolated from a Ni-Schiff-base complex. Hydrolysis conditions presented here are suitable for use when retention of acid-labile side-chain protecting groups is required. This technique may be adaptable to a variety of unnatural amino acid substrates.

Koruma Grupları aside dayanıksız Saklanmasına için uygun bir Ni-Schiff-Base hidrolizi Kompleksi kullanma Koşullar

Unnatural amino acids, amino acids containing side-chain functionalities not commonly seen in nature, are increasingly found in synthetic peptide ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

Heteromerik Protein Kompleksi-DNA Etkileşim Çalışmaları İçin Modifiye Maya-Bir Hibrid Sistem

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Here, we present a protocol to quantify and qualify each sphingomyelin species using multiple reaction monitoring and MS/MS/MS mode, respectively.

Sphingomyelin türler Sphingomyelin LC-MS iki kararlı Isotopically kullanarak tarafından için nicel ve nitel yöntemi etiketli

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Demir içeriği tesislerinde ölçme kolorimetrik yöntemi

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Eşleme metabolizma: doğrudan dokusunda laktat dehidrogenaz etkinliğini izleme

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

Sampling and Pretreatment of Tooth Enamel Carbonate for Stable Carbon and Oxygen Isotope Analysis

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and paleoenvironmental research from recent historical periods back to over 10 million years ago. Bulk approaches provide a representative sample for the period of enamel mineralization, while sequential samples within a tooth can track dietary and environmental changes during this period. While these methodologies have been widely applied and described in archaeology, ecology, and paleontology, there have been no explicit guidelines to aid in the selection of necessary lab equipment and to thoroughly describe detailed laboratory sampling and protocols. In this article, we document textually and visually, the entire process from sampling through pretreatment and diagenetic screening to make the methodology more widely available to researchers considering its application in a variety of laboratory settings.

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been used as a proxy for individual diet and environmental reconstruction. Here, we provide a detailed description and visual documentation of bulk and sequential tooth enamel sampling as well as pretreatment of archaeological and paleontological samples.

Örnekleme ve diş minesini Önarıtma kararlı karbon ve oksijen izotop analizi için karbonat

Stable carbon and oxygen isotope analysis of human and animal tooth enamel carbonate has been applied in paleodietary, paleoecological, and ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Ölçme ve oksijen Tüketim oranları tamamen yorumlama kafa kesimleri sinek

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Measuring Composition of CD95 Death-Inducing Signaling Complex and Processing of Procaspase-8 in this Complex

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (TRAIL-R1/R2). Stimulation of these receptors with their cognate ligands leads to the assembly of the death-inducing signaling complex (DISC). DISC comprises DR, the adaptor protein Fas-associated protein with death domain (FADD), procaspases-8/-10, and cellular FADD-like interleukin (IL)-1&#946;-converting enzyme-inhibitory proteins (c-FLIPs). The DISC serves as a platform for procaspase-8 processing and activation. The latter occurs via its dimerization/oligomerization in the death effector domain (DED) filaments assembled at the DISC. 
Activation of procaspase-8 is followed by its processing, which occurs in several steps. In this work, an established experimental workflow is described that allows the measurement of DISC formation and the processing of procaspase-8 in this complex. The workflow is based on immunoprecipitation techniques supported by western blot analysis. This workflow allows careful monitoring of different steps of procaspase-8 recruitment to the DISC and its processing and is highly relevant for investigating molecular mechanisms of extrinsic apoptosis.

Here, an experimental workflow is presented that enables the detection of caspase-8 processing directly at the death-inducing signaling complex (DISC) and determines the composition of this complex. This methodology has broad applications, from unraveling the molecular mechanisms of cell death pathways to the dynamic modeling of apoptosis networks.

CD95 Ölüme Neden Olan SinyalIzasyon Kompleksinin Bileşiminin Ölçülmesi ve Procaspase-8'in Bu Komplekste İşleniş

Extrinsic apoptosis is mediated by the activation of death receptors (DRs) such as CD95/Fas/APO-1 or tumor necrosis factor-related apoptosis-inducing ...