Dr. R. Howard Berg, Donald Danforth bitki Bilim Merkezi onun rehberliği için entegre mikroskobu tesis Müdürü teşekkür ediyoruz ve confocal mikroskobu ile yardımcı olur. Yazarlar hiçbir bildirmek için çıkar çatışması var.

1. Defensins Fluorophores ile etiketlerine göre
<ol>
	<li>Canlı hücre içinde Antimikrobiyal Özellikleri yanı sıra alımı ve yerelleştirme defensin, üzerinde en az etkisi bir fluorophore seçin. Not: optimum fluorophore seçerek belirli deneysel hedefler üzerinde bağlıdır. Spektral ve kimyasal özellikleri, photostability, boyutu ve fluorophore adet da düşünülmelidir.</li>
	<li>Etiket seti ticari satıcıları tarafından sağlanan etiketleme uygun bir peptid kullanarak seçili fluorophore ile defensin. Alternatif olarak, fluorophore etiketli defensins kimyasal sentez. Bu çalışmada, MtDef5 DyLight550 ile defensin etiket bir ticari etiketleme kullanarak kit üreticinin iletişim kuralı ve tetramethyl rodamine göre (TAMRA)-etiketli MtDef4 defensin kimyasal sentez ticari olarak. Not: Bu fluorophore peptid üzerinde en az etki sağlamak için N veya C terminal kalıntısı eklenebilir beri kimyasal olarak sentezlenmiş fluorophore öğesini peptidler (özellikle için kısa peptidler 50 amino asitler veya daha az) kullanılması tavsiye edilir, Antimikrobiyal aktivite. Üçten fazla disülfür bağları ile uzun peptidler sentezi meydan okuyor. Bu durumda, bu peptid kiti etiketleme uygun bir piyasada bulunan peptit kullanarak en uygun bir fluorophore ile etiketli önerilir.</li>
	<li>Test etiketli defensin in vitro antimikrobiyal etkinliği ve minimum inhibitör konsantrasyonu (MIC) araştırılması için mantarlar karşı belirlemek. Not: Bu çalışmada, DyLight550-MtDef5 ve TMR-MtDef4 mikrofon Neurospora crassa ve Fusarium graminearumkarşı tespit edilmiştir.</li>
</ol>
2. mantar kültürleri ve büyüme orta
<ol>
	<li>Vogel'ın agar orta21 içeren tüp eğim ve ışık altında oda sıcaklığında 5 gün kuluçkaya N. crassa hisse senedi kültüründen conidia aktarın.</li>
	<li>F. graminearum PH-1 baskı plakaları 5 gün için tam orta boy (CM)22 28 ºC de içeren kültür. Conidia üretimi için 5 gün eski kültür F. graminearum PH-1 4 (10 mm çap) fiş 50 mL carboxymethyl Selüloz (CMC) orta (15 g carboxymethyl selüloz, Maya özü, 0.5 g MgSO4.7 H2O, 1 g NH 1 g aşılamak. 4 YOK3ve 1 gr KH2PO4) ve kültür 180 rpm'de döner bir shaker 28 ºC 4-7 gün için.</li>
</ol>
3. hazırlık Conidial süspansiyon 
<ol>
	<li>F. graminearum conidial süspansiyon
	<ol>
		<li>Girdap F. graminearum sıvı kültür ve 2 mL microcentrifuge tüp içine 1,5 mL mantar kültürü filtrasyon malzemesi (örneğin Miracloth) iki kat ile filtreleme tarafından toplamak conidia. Conidial süspansiyon 13,226 x 2 min için bir microcentrifuge g hızı, santrifüj kapasitesi.</li>
		<li>Süpernatant atın ve 1 mL Pelet yıkamak için steril su ekleyin.</li>
		<li>13,226 x g hızı 2 dk. atma için bir microcentrifuge içinde conidial süspansiyon süpernatant santrifüj kapasitesi ve Pelet 1 mL 2 X SFM (sentetik mantar medya)23resuspend.</li>
		<li>Hafif bir mikroskop altında hemasitometre kullanarak conidia saymak.</li>
		<li>Conidial süspansiyon 105 conidia/ml 2 X SFM ile ayarlayın.</li>
	</ol>
	</li>
	<li>N. crassa conidial süspansiyon
	<ol>
		<li>Büyüyen kültür (5 gün eski) N. crassa Vogel'ın sıvı orta 2 mL içeren bir microcentrifuge tüp için bir aşı döngü kullanarak küçük bir miktar aktarın.</li>
		<li>Girdap conidial süspansiyon ve filtrasyon malzemesi yeni bir microcentrifuge tüp içine filtreden.</li>
		<li>Bir microcentrifuge 2 dk. atma için maksimum hızda conidial süspansiyon süpernatant santrifüj kapasitesi ve conidial Pelet 1 mL Vogel'ın sıvı orta resuspend.</li>
		<li>Hafif bir mikroskop altında hemasitometre kullanarak conidia saymak.</li>
		<li>Conidial süspansiyon 105 conidia/ml Vogel'ın sıvı orta ile ayarlayın.</li>
	</ol>
	</li>
</ol>
4. örnek hazırlama ve Confocal mikroskobu
<ol>
	<li>MtDef4 mantar hücrelere defensin, hücre altı yerelleştirme
	<ol>
		<li>Her conidial süspansiyon (105 conidia/mL), 50 µL 35 mm cam alt microwell yemekleri 10 mm microwell pipet. Conidia gözlenmesi için doğrudan bir sonraki adıma veya germlings hazırlık, pipet 50 µL N. crassa ve F. graminearum için conidia 35 mm kültür yemekleri devam etmek ve 3-6 h, oda sıcaklığında çimlenme izin.</li>
		<li>Her microwell yemeğin için Mik fluorescently etiketli defensin 50 µL ekleyin ve 2,5 h için kuluçkaya. Burada kullanılan fluorophore etiketli defensins (Adım 1.3) mikrofon N. crassa ve F. graminearum için 3 µM ve TMR-MtDef4 N. crassa ve F. graminearum MIC 1 µM ve 12 µM, anılan sıraya göre.</li>
		<li>Membran-seçici, 2 µL boya FM4-64 ekleyin (son konsantrasyonu: 5 mikron) kültür çanağı ve 30 dk için kuluçkaya ve hemen üzerinde görüntüleme için confocal mikroskop bağlamak.</li>
		<li>Beyaz ışık lazer (WLL) seçin. İki lazer kaynakları 488 nm ve 550 nm TMR-MtDef4 ve FM4-64 boya, sırasıyla heyecanlandırmak için. TMR-MtDef4 floresan 580-700 nm, algılamak ve FM4-64 boya 690-800 nm, algılamak. Not: confocal mikroskobu karanlık odada gerçekleştirin.</li>
	</ol>
	</li>
	<li>Zaman atlamalı görüntüleme MtDef5 defensin içselleştirilmesi
	<ol>
		<li>50 µL N. crassa ve F. graminearum conidial süspansiyon (105 conidia/mL), 35 mm cam alt microwell yemekleri 10 mm microwell pipet. 10 mm microwell kaplanmamış ve alt kısmında bir No 1.5 cam vardır. Conidia gözlem için doğrudan bir sonraki adıma geçin. Germlings gözlem için conidia 3-6 h mikroskobu ile devam etmeden önce oda sıcaklığında için kuluçkaya.</li>
		<li>WLL üzerinde açın. 550 lazer satırında seçin nm fluorophore etiketli defensins (Adım 1.3) burada kullanılan ve FM4-64 %1.00 yoğunluğu ile heyecanlandırmak ve karşılık gelen dedektörleri etkinleştirmek için. Burada kullanılan fluorophore etiketli defensins (Adım 1.3) 560-600 nm algılandı ve FM4 64dye 690-800 nm algılandı. Not: düşük lazer yoğunluğu yüksek lazer yoğunluğu canlı mantar hücreleri zarar verebilir gibi canlı hücre görüntüleme için kullanın.</li>
		<li>Microwell çanak mikroskobunun bağlayın. 10 x hedefleri başlangıçta kullanarak hücreleri bulmak, o zaman geçiş yapmak için 100 x / 1,44 petrol amaç için daha yüksek büyütme. Not: Tarama moduna xyzt (Z-yığın ve hızlandırılmış modları kombinasyonu), Z konumunu, Zoom, frekans görüntü yakalama, vs.</li></ol></li></ol>bir sonraki adıma devam etmeden önce.
		<li>Burada kullanılan fluorophore etiketli defensins (Adım 1.3) 50 µL 3 µM mikrofon ve membran-seçici boya FM4-64 (son konsantrasyonu 5 µM) 2 µL her microwell yemek için eklemek ve hızlandırılmış görüntüleme başlatın. Küçük ıslak filtreyi bildiri buharlaşma önlemek için microwell yemekleri alin. Görüntü yakalama sıklığı 3 dk 30 sn ve toplam süresi 2 saat 30 dk oldu. Not: Ekleme defensin ve montaj microwell tabak içinde mikroskop bölgenin ilgi bulanıklık sonra fluorophore. Bu nedenle, defensin ve fluorophore için microwell ekledikten sonra bölge ilgi refocused gecikme resim alma neden olabilir gerekir.</li>
		<li>Confocal mikroskop için tüm confocal görüntüsü kullanmak ve karanlık bir odada oda sıcaklığında mikroskobu yürütmek.</li>

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Yazarlar ifşa gerek yok.

Bu çalışmada, bir güvenilir canlı hücre görüntüleme metodoloji fluorescently etiketli antifungal defensins kullanımı ile Bu peptidler içselleştirilmesi mantar hücreleri içine Kinetik çalışmaya ve hücre altı hedefleri belirlemek için tanımlanmıştır. Bu yöntem defensins ve mantar hücreleri arasındaki etkileşimi dinamikleri geçici ve dağınık şekilde görselleştirmek için güçlü bir araçtır.
İçselleştirilmesi ve bitki defensins mantar hücrelerinde hücre...

Bitkiler mikrobiyal bitki patojenleri1karşı savunma için sofistike bir doğuştan gelen bağışıklık sistemi gelişmiştir. Onlar çok sayıda gen kodlanmış konak savunma peptidler sözde antimikrobiyal aktivite2hızlı. Gerçekten de, birçok Bu peptidler antimikrobiyal aktivitesi vitro3görüntüler. Defensins konak savunma peptidler bitki Krallık4en büyük gruplarından birini oluşturmaktadır. Bu sistein-zengin, Katyonik peptidler mantar karşı güçlü büyüme inhibitör etkinliği sergi ve oomycete patojenleri micromolar konsantrasyonları ve bu patojenler5,6karşı savunma ilk satırlardan birini temsil eder. Onların güçlü antifungal etkinlik nedeniyle, defensins hastalığı dayanıklı bitkiler üretmek için agribiotechnological uygulamalarında yararlanılabilir. Birkaç bitki defensins kurucu overexpression transgenik6sera ve alan testlerinde hastalık direnci artırmak için gösterilmiştir. Eylem bu antifungal peptidler (MOA) mekanizmaları tam olarak bitki koruma için etkili araçlar olarak potansiyellerini koşum için çözmek önemlidir. Ancak, bu bitki defensins MOA kötü anlaşılır. Geçerli kanıtlar onlar farklı MOA5,6,7,8sergi gösteriyor. Bazı defensins extracellularly mantarlar hareket ve belirli hücre duvarı/plazma membran ikamet organizmalardandır hedef, membran bütünlüğü bozabilir ve hücresel toksisite yolları9,10,11etkinleştirme. Son zamanlarda, ancak, mantar hücrelere translocate antifungal defensins bulunan12,13,14olmuştur. Bu defensins bazıları membran yerleşik biyoaktif fosfolipitler bağlamak oligomeric kompleksler oluşturur ve plazma membran15,16,17permeabilize. Böylece, bitki defensins MOA bazı yönlerini aydınlatılmamıştır. Ancak, bitki defensins büyük olasılıkla MOA henüz tespit edilmiş ve kapsamlı bir modeli olarak entegre olaylar karmaşık bir dizi içerir. Özellikle, anlayışımız Bu peptidler hücresel hedefler büyük bir boşluk kalır.
Mikroskopi teknolojiler ve yeni floresan problar gelişimi son gelişmeler ile canlı hücre görüntüleme teknikleri şimdi sık sık antimikrobiyal peptidler (Amper) MOA incelemek için kullanılır. Bu teknikler çoğunlukla morfolojisi antifungal peptidler etkilerini çözümlemek için istihdam immunolocalization, elektron mikroskobu, Atomik kuvvet mikroskobu veya x-ışını tomografi18, gibi yaygın olarak kullanılan yöntemleri tamamlayacak ve hücre duvarı bütünlüğü, değişiklikler hücre büyüme/dallanma desenleri, hem de plazma zarı permeabilization çalışmanın da dahil olmak üzere ve öldürmek mantar hücrelerinin büyümesini. Yine de, bu çalışmalar gerçekleştirmek yerine peptidler ile tedavi meydan defensin karşılık dinamik değişiklikleri izlemek için aynı hücrelerde Imaging hızlandırılmış sonra hücreleri belirli bir zaman noktasında Imaging'e sınırlı olmuştur. Son yıllarda fluorescently etiketli peptidler canlı hücre confocal mikroskobu kullanarak Imaging ile birlikte kullanımı gerçek zamanlı görselleştirme AMP-mikrop etkileşimleri dinamikleri sağlamıştır. Her ikisi de doğal olarak saflaştırılmış ve kimyasal olarak sentezlenmiş antifungal peptidler floresan etiketlerle öğesini (örneğin, DyLight, rodamine, BODIPY veya Alexa Fluor esaslı boyalar) ve doğrudan hücrelerle onların etkileşim sırasında hızlandırılmış tarafından gözlenen canlı hücre görüntüleme. Bunlar peptidler etiketli kullanımını önemli ölçüde onların MOA giriş, hücre altı yerelleştirme, hücre içi trafiği ve siteleri mantar önleyici eylem yaşam mantar hücreleri içinde modu dahil olmak üzere farklı yönleri anlayışımız artmıştır 18.
Son zamanlarda, çeşitli çalışmalar bitki defensins de dahil olmak üzere çeşitli antifungal peptidler yaşayan mantar hücreleri12,14,19,20tarafından içselleştirilmiş göstermiştir. Bu defensins büyük olasılıkla MOA etkileşim hücre içi hedefleri olan gerektirir. Son zamanlarda MtDef4 iki ascomycete Mantarlar, Neurospora crassa ve Fusarium graminearumdefensin bir bitkinin antifungal eylem bildirdin. MtDef4 mantar Hücre girişini ve bu mantar14hücre altı yerelleştirme için farklı yolları kullanmak için gösterildi. Tetramethyl rodamine (TAMRA) Bu çalışmada kullanılan kimyasal sentez-hayati floresan boyalar (membran seçici boya, FM4-64; hücre ölüm gazeteci boya, propidium iyodür membran permeant boya, SYTOX yeşil) ile birlikte MtDef4 etiketli ve metabolik inhibitörleri. Bu analizler MtDef4, onun mekanizmaları hücre içi ulaşım ve onun hücre altı hedefleri14içselleştirilmesi Kinetik gösterdi.
Burada, confocal mikroskobu kullanarak canlı hücre görüntüleme için bir protokol sunulmuştur. Protokol fluorescently etiketli peptidler bitki defensin mantar etkileşimleri, özellikle eğitim için hayati floresan boyalar, translocation yollar ile defensins mantar hücrelerinde hücre içi hedefleri ile birlikte kullanır.

<table>
	<tbody>
		<tr>
			<td>FM4-64 Dye</td>
			<td>Life Technologies</td>
			<td>T13320</td>
			<td></td>
		</tr>
		<tr>
			<td>DyLight 550 Antibody Labeling Kit</td>
			<td>Thermo Scientific</td>
			<td>84530</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Microwell Dishes</td>
			<td>Mat TeK</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Mira cloth</td>
			<td>EMD Millipore Corp</td>
			<td>475855-1R</td>
			<td></td>
		</tr>
		<tr>
			<td>SP8-X confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>FiJi</td>
			<td></td>
			<td>For Image analysis</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane</td>
			<td></td>
			<td>For Image analysis</td>
		</tr>
	</tbody>
</table>

live-cell imaging of fungal cells to investigate modes of entry and subcellular localization of antifungal plant defensins

Küçük sistein-zengin defensins bir ana savunma peptidler tüm tesislerinde mevcut en büyük grupları vardır. Birçok bitki defensins karşı geniş spektrumlu fungal patojenlerin güçlü vitro mantar önleyici faaliyet gösteren ve bu nedenle potansiyel transgenik antifungal ajanları olarak kullanılmak üzere. Bitki defensins hastalıkların kontrolü için tam potansiyelini koşum için kendi mekanizmaları eylem (MOA) aydınlatmak önemlidir. Gelişmiş mikroskobu teknikleri ortaya çıkmasıyla, canlı hücre görüntüleme bitki defensins antifungal MOA dinamiklerini anlamak için güçlü bir araç haline gelmiştir. Burada, bir temel confocal mikroskobu canlı hücre görüntüleme yöntemi hayati floresan boyalar ile birlikte iki fluorescently etiketli bitki defensins (MtDef4 ve MtDef5) kullanılarak açıklanmıştır. Bu teknik, gerçek zamanlı görüntüleme ve mantar hücreleri içine MtDef4 ve MtDef5 içselleştirilmesi dinamik olayların analizi sağlar. Önemlisi, bu tahlil içselleştirilmesi Kinetik, giriş modunu ve Bu peptidler hücre altı yerelleştirme dahil olmak üzere bilgi hazinesi oluşturur. Diğer hücre biyolojik aracın yanı sıra, bu yöntemler dinamikleri ve Bu peptidler MOA karmaşıklığı önemli anlayışlar hazırladık. Bu araçlar aynı zamanda farklı mantarlar karşı Bu peptidler MOA karşılaştırmak için kullanılabilir.

Canlı hücre görüntüleme gerçekleştirildiği parça ve içselleştirilmesi ve iki defensins, MtDef4 ve MtDef5, Medicago truncatula; dan hücre altı yerelleştirme karşılaştırmak için mantar hücrelerinde. TMR-MtDef4 kimyasal sentez MtDef5 Dylight550 ile etiketli iken (Dylight550-MtDef5). Conidia membran seçici boya FM4-64 ile birlikte her iki defensin ile inkübe. TMR-MtDef4 F. graminearumiçin karşılaştırıldığında N. crassa farklı ticaret ...

Watch this Scientific Journal Video about Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins at JoVE.com

Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Bitki defensins patojenlere karşı bitki savunmasında önemli bir rol oynamaktadır. Etkili eylem (MOA) onların mod anlama antifungal ajanları olarak antifungal Bu peptidler kullanımı önemlidir için. Burada, Bu peptidler MOA kritik yönlerini incelemek için canlı hücre görüntüleme yöntemi açıklanmıştır.

Canlı hücre görüntüleme modları giriş ve Antifungal bitki Defensins hücre altı yerelleştirme araştırmak için mantar hücrelerinin

Small cysteine-rich defensins are one of the largest groups of host defense peptides present in all plants. Many plant defensins exhibit potent in vitro ...

live-cell-imaging-fungal-cells-to-investigate-modes-entry-subcellular

Research

JoVE Journal

Biology

6.6K Görüntüleme.  Donald Danforth Plant Science Center. Bitki defensins patojenlere karşı bitki savunmasında önemli bir rol oynamaktadır. Etkili eylem (MOA) onların mod anlama antifungal ajanları olarak antifungal Bu peptidler kullanımı önemlidir için. Burada, Bu peptidler MOA kritik yönlerini incelemek için canlı hücre görüntüleme yöntemi açıklanmıştır.

Video: Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Canlı hücre Görüntüleme ve Kantitatif Analiz Embriyonik Epitel Hücreleri Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Biyoloji

Fluorescence-based Measurement of Store-operated Calcium Entry in Live Cells: from Cultured Cancer Cell to Skeletal Muscle Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to replenish depleted endoplasmic reticulum (ER) or sarcoplasmic reticulum (SR) Ca2+ stores1,2. Since Ca2+ is a ubiquitous second messenger, it is not surprising to see that SOCE plays important roles in a variety of cellular processes, including proliferation, apoptosis, gene transcription and motility. Due to its wide occurrence in nearly all cell types, including epithelial cells and skeletal muscles, this pathway has received great interest3,4. However, the heterogeneity of SOCE characteristics in different cell types and the physiological function are still not clear5-7.
The functional channel properties of SOCE can be revealed by patch-clamp studies, whereas a large body of knowledge about this pathway has been gained by fluorescence-based intracellular Ca2+ measurements because of its convenience and feasibility for high-throughput screening. The objective of this report is to summarize a few fluorescence-based methods to measure the activation of SOCE in monolayer cells, suspended cells and muscle fibers5,8-10. The most commonly used of these fluorescence methods is to directly monitor the dynamics of intracellular Ca2+ using the ratio of F340nm and F380nm (510 nm for emission wavelength) of the ratiometric Ca2+ indicator Fura-2. To isolate the activity of unidirectional SOCE from intracellular Ca2+ release and Ca2+ extrusion, a Mn2+ quenching assay is frequently used. Mn2+ is known to be able to permeate into cells via SOCE while it is impervious to the surface membrane extrusion processes or to ER uptake by Ca2+ pumps due to its very high affinity with Fura-2. As a result, the quenching of Fura-2 fluorescence induced by the entry of extracellular Mn2+ into the cells represents a measurement of activity of SOCE9. Ratiometric measurement and the Mn+2 quenching assays can be performed on a cuvette-based spectrofluorometer in a cell population mode or in a microscope-based system to visualize single cells. The advantage of single cell measurements is that individual cells subjected to gene manipulations can be selected using GFP or RFP reporters, allowing studies in genetically modified or mutated cells. The spatiotemporal characteristics of SOCE in structurally specialized skeletal muscle can be achieved in skinned muscle fibers by simultaneously monitoring the fluorescence of two low affinity Ca2+ indicators targeted to specific compartments of the muscle fiber, such as Fluo-5N in the SR and Rhod-5N in the transverse tubules9,11,12.

The extent of store-operated Ca2+ entry (SOCE) can be monitored using fluorescent Ca2+ indicators. Mn2+ quenching of such indicators assays SOCE in cultured cells and skeletal muscle fibers. A technique allowing spatial and temporal resolution of SOCE by confocal imaging of mechanically skinned muscle fibers is also described.

Canlı hücreler Mağaza çalışan Kalsiyum Giriş Floresans tabanlı Ölçümü: Kültür Kanseri Hücre gelen İskelet Kası Fiber

Store operated Ca2+ entry (SOCE), earlier termed capacitative Ca2+ entry, is a tightly regulated mechanism for influx of extracellular Ca2+ into cells to ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Live Cell Imaging of Early Autophagy Events: Omegasomes and Beyond

Autophagy is a cellular response triggered by the lack of nutrients, especially the absence of amino acids. Autophagy is defined by the formation of double membrane structures, called autophagosomes, that sequester cytoplasm, long-lived proteins and protein aggregates, defective organelles, and even viruses or bacteria. Autophagosomes eventually fuse with lysosomes leading to bulk degradation of their content, with the produced nutrients being recycled back to the cytoplasm. Therefore, autophagy is crucial for cell homeostasis, and dysregulation of autophagy can lead to disease, most notably neurodegeneration, ageing and cancer.	
	Autophagosome formation is a very elaborate process, for which cells have allocated a specific group of proteins, called the core autophagy machinery. The core autophagy machinery is functionally complemented by additional proteins involved in diverse cellular processes, e.g. in membrane trafficking, in mitochondrial and lysosomal biology. Coordination of these proteins for the formation and degradation of autophagosomes constitutes the highly dynamic and sophisticated response of autophagy. Live cell imaging allows one to follow the molecular contribution of each autophagy-related protein down to the level of a single autophagosome formation event and in real time, therefore this technique offers a high temporal and spatial resolution.	
	Here we use a cell line stably expressing GFP-DFCP1, to establish a spatial and temporal context for our analysis. DFCP1 marks omegasomes, which are precursor structures leading to autophagosomes formation. A protein of interest (POI) can be marked with either a red or cyan fluorescent tag. Different organelles, like the ER, mitochondria and lysosomes, are all involved in different steps of autophagosome formation, and can be marked using a specific tracker dye. Time-lapse microscopy of autophagy in this experimental set up, allows information to be extracted about the fourth dimension, i.e. time. Hence we can follow the contribution of the POI to autophagy in space and time.

Time-lapse microscopy of fluorescently labeled autophagy markers allows monitoring of the dynamic autophagy response with high temporal resolution. Using specific autophagy and organelle markers in a combination of 3 different colors, we can follow the contribution of a protein to autophagosome formation in a robust spatial and temporal context.

Erken Otofaji Etkinlikler Hücre Görüntüleme Canlı: Omegasomes ve Ötesi

Autophagy is a  cellular response triggered by the lack of nutrients, especially the absence of  amino acids. Autophagy is defined by the formation of ...

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM.
Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells.
The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Scanning Electron Microscopy SEM Protocols for Problematic Plant, Oomycete, and Fungal Samples

Problems in the processing of biological samples for scanning electron microscopy observation include cell collapse, treatment of samples from wet microenvironments and cell destruction. Low-cost and relatively rapid protocols suited for preparing challenging samples such as floral meristems, oomycete cysts, and fungi (Agaricales) are compiled and detailed here.

Sorunlu Tesisi, Oomiçet ve fungal Örnekleri için Elektron Mikroskobu (SEM) Protokoller tarayarak

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

pHluorin etiketli Reseptör kullanarak Subselüler Yerelleşme ve Ticareti Monitör

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

Live-cell Imaging of Platelet Degranulation and Secretion Under Flow

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the formation of thrombi that occlude the vasculature and injure organs, with life-threatening consequences. This motivates scientific research on platelet function and the development of methods to track cell-biological processes as they occur under flow conditions.
A variety of flow models are available for the study of platelet adhesion and aggregation, two key phenomena in platelet biology. This work describes a method to study real-time platelet degranulation under flow during activation. The method makes use of a flow chamber coupled to a syringe-pump setup that is placed under a wide-field, inverted, LED-based fluorescence microscope. The setup described here allows for the simultaneous excitation of multiple fluorophores that are delivered by fluorescently labeled antibodies or fluorescent dyes. After live-cell imaging experiments, the cover glasses can be further processed and analyzed using static microscopy (i.e., confocal microscopy or scanning electron microscopy).

This work describes a fluorescence microscopy-based method for the study of platelet adhesion, spreading, and secretion under flow. This versatile platform enables the investigation of platelet function for mechanistic research on thrombosis and hemostasis.

Akım altında Trombosit Degranülasyonu ve Salgılamanın Canlı Hücreli Görüntüleme

Blood platelets are essential players in hemostasis, the formation of thrombi to seal vascular breaches. They are also involved in thrombosis, the ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

Hücre Altı Yapıların Süper Çözünürlüklü Canlı Hücre Görüntülemesi

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...

Live-cell Imaging of Single-Cell Arrays (LISCA) - a Versatile Technique to Quantify Cellular Kinetics

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high throughput. In general, the acquisition of single-cell time courses from cultured cells is hampered by cell motility and diversity of cell shapes. Adhesive micro-arrays standardize single-cell conditions and facilitate image analysis. LISCA combines single-cell microarrays with scanning time-lapse microscopy and automated image processing. Here, we describe the experimental steps of taking single-cell fluorescence time courses in a LISCA format. We transfect cells adherent to a micropatterned array using mRNA encoding for enhanced green fluorescent protein (eGFP) and monitor the eGFP expression kinetics of hundreds of cells in parallel via scanning time-lapse microscopy. The image data stacks are automatically processed by newly developed software that integrates fluorescence intensity over selected cell contours to generate single-cell fluorescence time courses. We demonstrate that eGFP expression time courses after mRNA transfection are well described by a simple kinetic translation model that reveals expression and degradation rates of mRNA. Further applications of LISCA for event time correlations of multiple markers in the context of signaling apoptosis are discussed.

Live-cell Imaging of Single-Cell Arrays LISCA - a Versatile Technique to Quantify Cellular Kinetics

We present a method for the acquisition of fluorescence reporter time courses from single cells using micropatterned arrays. The protocol describes the preparation of single-cell arrays, the setup and operation of live-cell scanning time-lapse microscopy and an open-source image analysis tool for automated preselection, visual control and tracking of cell-integrated fluorescence time courses per adhesion site.

Tek Hücreli Dizilerin Canlı Hücre Görüntülemesi (LISCA) - Hücresel Kinetiği Ölçmek için Çok Yönlü Bir Teknik

Live-cell Imaging of Single-Cell Arrays (LISCA) is a versatile method to collect time courses of fluorescence signals from individual cells in high ...