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1. hücre kültürü edinme izleme
<ol><li>DMEM + (örneğin, GlutaMAX) glutamin HeLa hücreleri büyümek orta % 10 (v/v) fetal buzağı ısı inaktive serum ve % 1 penisilin ve streptomisin ile desteklenmiştir.</li>
	<li>6-iyi cam alt kültür pilakalar ve fibronektin (25 µg/mL) 1 h için kat. O zaman iyi başına 2 x 104 hücre tohum.</li>
	<li>Satın alma sırasında orta 3 günde değiştirin.</li>
	<li>Hızlandırılmış edinmesinin video mikroskop (ticari olarak mevcut) objektif ücretsiz kullanın. Not: Bu objektif-Alerjik Hesaplamalı görüntüleme tekniği üzerinde Özcan vd tarafından açıklandığı gibi temel alır 16 bir kuluçka 37 ° C12,15kontrollü bir sıcaklık sürekli izleme gerçekleştirmek için güncellenmiştir. 1,67 µm bir piksel aralığı ve görüntüleme 6.4 x 4.6 mm2lik bir tamamlayıcı metal oksit yarı iletkeni (CMOS) görüntü algılayıcı bulunuyor. Birden çok dalga boyu aydınlatma kırmızı, yeşil ve mavi aydınlatma sağlayan bir multichip ışık yayan diyot (LED) aygıtı tarafından sağlanır. 636, 521 ve 452 dalga boylarında ortalanır nm sırasıyla, spektral bant genişliği 25, 45 ve 25 ile nm anılan sıraya göre. Kırmızı-yeşil-mavi (RGB) LED hücreleri yaklaşık 5 cm uzaklıkta bir 150 µm iğne deliği yukarıda yer alır. Ölçülen yakın LED ışık güç gibi düşük 10 µW at her dalga boyu ve aydınlatma zaman edinme başına yalnızca bir saniye. Bu nedenle, hücre kültürlerinde objektif-ücretsiz video mikroskop altında gözlendiğinde fotoğraf-toksisite sorun bekleniyor.</li>
	<li>CMOS sensör (şekil 1) ile temas koymak hücre kültür kap koymak. Objektif-Alerjik edinme kalitesi için önemlidir alt cam coverslip ile bir kapsayıcı kullanın. Plastik kaplar-ebilmek da var kullanılmış ama objektif-Alerjik edinme görme duyusuyla ilgili kalitesiz bu kapları sayesinde düşer.</li>
	<li>Hızlandırılmış edinme ve holografik imar yapan video objektif-Alerjik mikroskop edinme yazılım (ticari olarak mevcut) ile kontrol. Yazılım arayüzü kullanıcı tarafından girilmesi gerekir kare hızı, deneme ve hücre kültürü, yani yapışık hücreleri veya yüzen hücreleri türünü süresi parametrelerdir.</li>
	<li>Satın alma yazılım giriş parametrelerini ayarlamak. Her 10 dakikada bir edinme küme ve hücre kültür tipi 'yapışkan' kare hızı ayarlayın. Not: güvenilir bir hücre çözümlenmesi için elde edilen veri kümesi boyutunu hala makul olmakla birlikte izleme sağlar bir alım her 10 dakikada bir kare hızı iyi bir uzlaşma gibidir. En hızlı kare hızı bu objektif-Alerjik mikroskobu kurulum ile ulaşılabilir bir 5 dakikada değil. Daha fazla kare hızını artırarak aşırı ısınma kültür hücrelerdeki canlılık etkileyebilecek CMOS sensörünün sonuçlanır.</li>
	<li>Hızlandırılmış edinme başlatmak ve holografik imar algoritması (ticari olarak mevcut) otomatik olarak hücre kültürü faz görüntüsünü elde etmek için objektif ücretsiz satın alma etkin olur. Holografik imar algoritması bir çoklu dalga boyu faz alma algoritması17 tarihinde dayanmaktadır ve RGB yeniden oluşturulan faz görüntüyü aralığında sağlar [-π, + π] 29.4 mm2görüş büyük bir alan üzerinde tek her hücre için. Bir objektif içermeyen satır içi Holografi ince bir örnek aydınlatmak için ilkesidir (z = 0) ile bir düzlem dalga (Toplam genlik 1 normalleştirme sonra) ve bir CMOS sensör üzerinde sonraki kırınım yoğunluğu görüntü kısa mesafe z algılamak için Z, sonra genellikle 1 mm = örnek. Bizim kurulum, aydınlatma sırayla 3 boyları için açık olduğundan (λ10,450 µm, λ2= 0.540 µm, λ3= 0.647 µm =) üç kırınım desen aynı örnek ölçmek için.</li>
</ol>2. hücre Kültür veri analizi
<ol><li>Hücre-izleme için Trackmate algoritması, tek parçacıklar18otomatik izleme için bir açık kaynak Fiji eklenti kullanın. İlk olarak, yük tam hızlandırılmış edinme Fiji (yük görüntü sırası komutu). Genellikle 400 RGB çerçeveler 29,7 MB özellikleri, veri kümeleri, büyüklüğü nedeniyle onları 8-bit biçimde yük daha hızlı olur. Sonraki Trackmate Fiji eklenti birkaç aşamada hücre izleme algoritması, yani bir hücre algılama sahne, bir hücre izci sahne ve hücre tespitlerde ve hesaplanan parça için uygulanan çeşitli filtreler aracılığıyla kullanıcıya gösterir. Her aşamada algoritmalar yapılandırmak ve sonuçları görüntülemek böylece hemen gerekirse, bir kolayca ileri geri ayarları yeniden düzenlemek için gidebilirsiniz. Trackmate arabiriminin çeşitli menüler Şekil 2 ' HeLa hücreleri deneme için kullanılan gerçek ayarları ile gösterilir.</li>
	<li>(Bkz: Şekil 2) takip olarak satın alma yazılım giriş parametrelerini ayarlamak. Tahmini blob çapı 15 piksel, 0,25 dedektörü eşik, bağlantı maksimum mesafe 15 piksel, gap-kapanış en fazla uzaklık 15 piksel ve 3.5 parçalardaki noktaların sayısını ayarlayın. Not: Bu ayarlar belli ki bir deneyden başka bir '(burada 1,67 µm piksel cinsinden verilen) hücre boyutu karşılık gelen özellikle tahmini blob çapı' farklı olacaktır. Aynı remark '(burada 1,67 µm piksel cinsinden verilen) iki ardışık çerçeveler içinde bir hücre tarafından hangi hesap için maksimum mesafe yolculuk bağlantı maksimum mesafe' geçerlidir. En uygun değerini hücre hareketliliği aynı zamanda hücre yoğunluğuna bağlı olacaktır.</li>
	<li>Hücre izleme işleminin sonunda sonuçları üç metin dosyası şeklinde oluşturma ('Analiz' düğme, bkz: Şekil 2). 'Noktalar parça istatistikleri' adlı en yararlı dosya (x, y) ilgili konumlarını satın alma, onların çerçeve numarası ve onların parça numarası ile algılanan tüm hücreleri listelendiği bir tablo oluşur. Bu bilgiler temelinde hücre segmentasyon gerçekleştirmek ve hücre adanmış algoritmaları (bkz: ek kod dosyaları) kullanarak bölümler algılamak. Diğer iki dosya 'Parça istatistikleri bağlantılar' adlı ve 'İzlemek istatistikleri' ve ilgili parça, örneğin parça sürelerini, algılanan boşluklar sayısı ile tüm sonuçlar içerir, izlemek ilk çerçeve, vb.</li>
	<li>Hücre segmentasyon algoritma kullanın (bkz: ek kod dosyasını otomatik olarak yeniden oluşturulan faz görüntüden hücre morfolojisi açıklayan çeşitli ölçümler ayıklamak için). Bu ölçümler hücre yüzey alanı (S), hücre Binbaşı eksen uzunluğu (M), hücre küçük eksen uzunluğu (m) ve hücre en boy oranı (L/m) dir. Objektif-Alerjik mikroskobu kurtarılan yoğunluğu ve15daha önce tartııldıı gibi numune katman kalınlığı orantılı aşamasıdır.</li>
	<li>Hücre morfolojik özelliklere ek olarak, nicel hücre kuru kitle (CDM) ölçümleri yeniden oluşturulan faz13,19,20 ayıklayın. <img alt="Equation 1" src="/files/ftp_upload/56580/56580eq1v3.jpg">EQ (1) yeniden oluşturulan faz kayması19, λ dalga boyu ve α nerede φ(x,y) = 1.8 x 10-4 m3/kg kitle1, Kuru için Kırılma indisi varyasyonu ilgili belirli Kırılma indisi 19.</li>
	<li>Hücre Kırılma indisi ile bu kültür ortamının arasındaki fark bir tahmini kullanarak hücre kalınlığı değerlendirin. Hücre kalınlığı h(x,y) ve ölçülen faz kayması ilişkisi20tarafından verilir: <img alt="Equation 2" src="/files/ftp_upload/56580/56580eq2v3.jpg">EQ (2,1) <img alt="Equation 3" src="/files/ftp_upload/56580/56580eq3v3.jpg">EQ (2,2) Δn(x,y) = nhücre (x,y) - nOrta (x,y), hücre ve kültür medya arasındaki Kırılma indisi fark. Aşağıda, Δ sabit bir değer kabuln 0,025 =, hangi HeLa hücre çekirdeği içinde ölçülen Kırılma indisi üzerinden tahmin ediliyor (n = 1.35511) ve fosfat tamponlu tuz çözeltisi (PBS) kültür ortamının (n 1.33 =). Her ne kadar hücre Kalınlık değeri yalnızca bir varsayıma dayalı olarak göstergesidir, göreli varyasyonları anlamlı ve sayesinde bunun bölünen hücreler algılamak için kullandı.</li>
	<li>Özel algoritması kullanın (hücre bölünmeler oluşumunu algılayan ve hücre bölünmesi algılamak için tespit edilen hücre bölümler için ilişkili hücre parçaları ayıklamak için bkz: ek kod dosyası) hücreleri ile ölçülen kalınlık 8 µm büyük kontrol yeni bir hücre yakından olarak görünüp görünmeyeceğini belirtir uzay ve zaman o zaman ilk, tanımlanır. Bu şekilde, hücre bölünmeler algılanması iki sağlam kriterlere dayanır. Alternatif ve hücre bölünmesi algılama için daha ayrıntılı yöntemleri için lensfree hızlandırılmış edinme21,22,23uygulanabilir.</li>
</ol>

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B., Kafri, R., Kirschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+being+the+right+(cell)+size.">On being the right (cell) size.</a> Science. 348 (6236), 1245075 (2015).</li><li>Mathieu, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+lens-free+imaging+of+cell+migration+in+diverse+physical+microenvironments.">Time-lapse lens-free imaging of cell migration in diverse physical microenvironments.</a> Lab Chip. 16 (17), 3304-3316 (2016).</li></ol>

Yazarlar ifşa gerek yok.

Bu yazıda, bu objektif-ücretsiz video mikroskobu içinde bir kuluçka hücreleri binlerce Kinetik yakalamak için kullanılabileceğini göstermektedir. Genel yöntembilim tanımlamak için HeLa hücreleri kültür 2,7 gün hızlandırılmış edinimi Standart hücre izleme algoritmalarıyla nasıl çözümlenebilir açıkladı. 2.2 x 106 hücre ölçümleri ve 10,584 hücre döngüsü parça içeren bir veri kümesi sonucudur. Satın almalar Hela hücreleri nispeten büyük bir hücre hücre mesafe ile bir kültür olarak gerçekleştirilmiştir (hücre yoğunluğu < 500 hücre/mm2) ve hücre bir diskle birlikte yaklaşık morfolojisi. Bu iki özellik büyük bir veri toplamak mümkün kılan bir otomatik olarak hücre izleme analiz uygulanmasının kolaylaştırılması. Diğer hücre hatları ile daha küçük hücre hücre mesafe veya daha karmaşık türleri morfoloji ile izlemek daha zor olacak ve sonraki veri kümelerini daha küçük olur.
Bu yaklaşım birçok çalışma için örneğin hücre çoğalması ve hücre boyutu çalışmaları için ilginç olduğunu düşünüyoruz. İkinci araştırma önemli bir alanı temel Biyolojide vardır ve birçok soru cevapsız kalır gibi güzel bir inceleme Ginzberg vd tarafından ele 29. bu çalışmada önerilen iletişim kuralı böylece önemli ölçütleri ölçmek için bir yol gerekli için Hücre proliferasyonu çalışmaları, yani hücre döngüsü süresi, hücre kuru kitle, hücre döngüsü ve hücre başlangıcı arasındaki sonunda kuru kitle oranı sağlar Kuru kitle büyüme oranı. O-ebilmek üretmek çok sayıda parça, her biri birkaç saat süren hücre çoğalması çalışmanın yanı sıra, bu yöntem hücre göç, incelenmesi için ilgi olacaktır. Mevcut protokolü en son14,30 hücre parça binlerce edinme daha 10 h sırasında ilk kez göstererek gider.
Sonuç olarak, etiket içermeyen hücreleri binlerce kültür birkaç gün içinde aynı anda izlemek için izin veren bir kullanımı kolay lens-Alerjik tekniği geliştirdik.

Boyut ve hücre hücre birkaç hücre döngüsü boyunca kültürlü memeli hücreleri izleme ve doğru ölçme kuru kütlesi olduğunu zor bir görev. Birkaç etiket içermeyen optik teknikleri bu görev1,2gerçekleştirmek edebiliyoruz: faz kayması Interferometry3, dijital holografik mikroskobu (DHM)4,5,6, 7, quadriwave yanal kesme Interferometry8,9 ve nicel faz tomografi10,11. Bu yöntemler memeli hücrelerinin hücre döngüsü anlayış içine birçok yeni anlayışlar açmıştır. Ancak nadiren algoritmaları izleme otomatik hücre ile birleştiğinde ve onların üretilen iş ne zaman ölçme sınırlı kalır kitle yörüngeleri1 hücre (N < 20 sırasıyla3,4,5 , 6). dolayısıyla bir roman optik yöntemi hücre kitle yörüngeleri büyük istatistikleri ile ölçmek için gereklidir (N > 1000).
Bu yazıda, biz aynı anda hücreleri doğrudan kuluçka makinesi içinde binlerce resim ve tek hücre ölçümleri tek hücre döngüsü parça binlerce boyunca ölçmek için objektif-ücretsiz video mikroskobu yeteneğini göstermek. Objektif-Alerjik mikroskobu mi nicel bir aşama aşama görüntü yoğun paketlenmiş hücre edinimi üzerinde görüş çok büyük bir alan sağlayan teknik görüntüleme (genellikle birkaç on mm2, burada 29.4 mm2)12,13 ,14,15. Tek hücre düzeyinde çeşitli ölçümler belirlenir, örneğin, hücre alanı, cep kuru kitle, hücre kalınlığı, hücre Binbaşı eksen uzunluğu ve en boy oranı12,15, her görüntü hücre. Sonra bir hücre izleme algoritma uygulayarak, bu özellikler tek her hücre için deneme süresi14,15bir fonksiyonu olarak çizilebilir. Ayrıca, hücre bölünmeler oluşumunu hücre parça olarak algılayarak, ilk hücre kuru kitle (hemen sonra hücre bölünmesi), son hücre kuru kitle (hemen önce hücre bölünmesi) ve hücre gibi diğer önemli bilgileri ayıklamak mümkün olduğunu döngüsü süresi, Yani, iki ardışık tümen15arasındaki süre. Bu ölçümler ile çok iyi istatistikler hesaplanabilir (N > 1000) geniş görüş alanı genellikle 200-10.000 hücreleri tek bir objektif-Alerjik kazanım analizi sağlayacak bu yana.
Objektif-ücretsiz video mikroskobu dayalı bu yöntemi açıklamak için protokolü için 2,7 gün bir HeLa hücre kültürü ölçmek ve izlemek için açıklar. Veri analizi çok dalga boyu holografik yeniden yapılanma, hücre izleme, hücre segmentasyon ve hücre bölünmesi algoritmaları dayalı dört adımlı bir işlemdir. Burada Standart hücre izleme algoritmaları ile uyumlu Uzaysal çözünürlük ve bu objektif-ücretsiz video mikroskobu Kur'a elde nispeten hızlı kare hızı (bir edinme her 10 dakikada bir) gösterilir. Bu veri kümesinin tam analiz tam hücre döngüsü 10,584 hücre parça ölçüm sonuçları.
Özet olarak, objektif-ücretsiz video mikroskobu otomatik olarak etiketlenmemiş, eşitlenmemiş binlerce ve deney başına değiştirilmemiş hücre izlemek için güçlü bir araçtır; birkaç hücre döngü üzerinde izlenen her hücre. Bizim ölçümleri böylece birkaç hücre parametreleri, ama daha da önemlisi, arası hücre değişkenlik hücrelerinin büyük bir nüfus üzerinden ortalama değerini sağlar.

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			<td height="20" style="height:20px;width:231px;">Cytonote lens-free video microscope&#160;</td>
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			<td height="20" style="height:20px;">Horus acquisition software</td>
			<td>Iprasense</td>
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			<td></td>
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			<td height="20" style="height:20px;">6-well glass bottom culture plates</td>
			<td>MatTek corporation</td>
			<td>Part No: P06G-0-14-F&#160;</td>
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			<td height="20" style="height:20px;">DMEM + GlutaMAX medium&#160;</td>
			<td>Gibco</td>
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			<td></td>
		</tr>
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			<td height="20" style="height:20px;">heat-inactivated fetal calf serum&#160;</td>
			<td>Eurobio</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">penicillin and streptomycin&#160;</td>
			<td>Gibco</td>
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			<td height="20" style="height:20px;">Fibronectin&#160;</td>
			<td>Sigma Aldrich</td>
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			<td height="20" style="height:20px;">Matlab, image processing toolbox&#160;</td>
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lens-free video microscopy for the dynamic and quantitative analysis of adherent cell culture

Burada, biz o objektif-ücretsiz video göstermek mikroskobu hücreleri doğrudan kuluçka makinesi içinde binlerce Kinetik aynı anda yakalamak için bize sağlar ve birkaç hücre döngüsü boyunca tek hücreleri ölçmek ve izlemek mümkündür. Biz 2,7 gün bir HeLa hücre kültürü ölçmek ve izlemek için kullanılan tam iletişim kuralı tanımlamak. İlk olarak, hücre kültürü edinme objektif-ücretsiz video mikroskopla gerçekleştirilir ve sonra verileri bir dört adımlı süreç aşağıdaki analiz edilir: Çoklu dalga boyu holografik yeniden yapılanma, hücre-izleme, hücre segmentasyon ve hücre bölünmesi algılama algoritmalar. Sonuç olarak, bir veri kümesi 10.000 hücre döngüsü parça ve 2 x 10'dan fazla6 hücre morfolojik ölçümler daha fazla featuring toplamak mümkün olduğunu gösteriyor.

Watch this Scientific Journal Video about Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture at JoVE.com

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Objektif-ücretsiz video mikroskobu hücre kültürleri doğrudan kuluçka makinesi içinde izlemek sağlar. Burada biz almak ve kültürlü HeLa hücreleri 2,7 gün uzun edinimi çözümlemek için kullanılan tam iletişim kuralı tanımlamak, 2.2 x 10 bir veri kümesi için önde gelen6 ölçümleri tek hücre morfolojisi ve 10584 hücre döngüsü izler.

Dinamik için objektif-ücretsiz Video mikroskobu ve kantitatif analiz yapisan hücre kültürü

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

lens-free-video-microscopy-for-dynamic-quantitative-analysis-adherent

Nanyang Technological University

Research

JoVE Journal

Biology

9.4K Görüntüleme.  Université Grenoble Alpes. Objektif-ücretsiz video mikroskobu hücre kültürleri doğrudan kuluçka makinesi içinde izlemek sağlar. Burada biz almak ve kültürlü HeLa hücreleri 2,7 gün uzun edinimi çözümlemek için kullanılan tam iletişim kuralı tanımlamak, 2.2 x 10 bir veri kümesi için önde gelen6 ölçümleri tek hücre morfolojisi ve 10584 hücre döngüsü izler.

Video: Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biyoloji

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

Video Biyoinformatik İnsan Embriyonik Kök Hücre Colony Büyüme Analizi

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenic Testi: yapışkan Hücreler

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Fisyon Maya Kantitatif Canlı Hücre Floresan mikroskopi Analizi

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Time-lapse video Mikroskop ile Hücrelerinin Rastgele Göç Kantitatif Analiz

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

Kantitatif Spor Analizi İş Akışı

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12.
Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. With commercially available digital image analysis applications, we can readily obtain statistical information about autophagosome and lysosome number, size, distribution, and degree of colocalization from any imaged cell. This information allows us to precisely track the progress of autophagy in living cells and enables our continued investigation into the role of autophagy in cancer chemotherapy.

Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autolysosomes.

Gelişmiş 3D Floresan Mikroskobu kullanılarak Otofaji Kantitatif Analiz

Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Periferik Kök Hücre Göç Analizi Fare Cremaster Muscle Mikrosirkülasyon Intravital Mikroskopi

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP Mikroskopisi ile Gap Birleşimine Bağlı MiRNA Transferinin Analizi

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of clustering. Here, we describe an imaging approach to determine the average oligomeric state of mEGFP-tagged-receptor homocomplexes in the membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis. N&#38;B is a method similar to fluorescence-correlation spectroscopy (FCS) and photon counting histogram (PCH), which are based on the statistical analysis of the fluctuations of the fluorescence intensity of fluorophores diffusing in and out of an illumination volume during an observation time. In particular, N&#38;B is a simplification of PCH to obtain information on the average number of proteins in oligomeric mixtures. The intensity fluctuation amplitudes are described by the molecular brightness of the fluorophore and the average number of fluorophores within the illumination volume. Thus, N&#38;B considers only the first and second moments of the amplitude distribution, namely, the mean intensity and the variance. This is, at the same time, the strength and the weakness of the method. Because only two moments are considered, N&#38;B cannot determine the molar fraction of unknown oligomers in a mixture, but it only estimates the average oligomerization state of the mixture. Nevertheless, it can be applied to relatively small time series (compared to other moment methods) of images of live cells on a pixel-by-pixel basis, simply by monitoring the time fluctuations of the fluorescence intensity. It reduces the effective time-per-pixel to a few microseconds, allowing acquisition in the time range of seconds to milliseconds, which is necessary for fast oligomerization kinetics. Finally, large cell areas as well as sub-cellular compartments can be explored.

We describe an imaging approach for the determination of the average oligomeric state of mEGFP-tagged-receptor oligomers induced by ligand binding in the plasma membrane of living cells. The protocol is based on Total Internal Reflection Fluorescence (TIRF) microscopy combined with Number and Brightness (N&#38;B) analysis.

Toplam İç Yansıma Floresan Mikroskobu Ile Canlı Hücrelerdeki Hücre Yüzey Reseptörlerinin Oligomerizasyon Dinamiği Sayı ve Parlaklık Analizi ile Birlikte

Despite the importance and ubiquity of receptor oligomerization, few methods are applicable for detecting clustering events and measuring the degree of ...