This research is supported by funding from CBCF (to ZW).

1. Canlı Görüntüleme Hücreleri hazırlanması <ol><li> Hücre kültürü ve tohum <ol><li> Dosetaksel (MCF-7 TXT) ve dirençli olmayan ana hücre dizisi (MCF-7 CC) karşı direnç için seçilir MCF-7 göğüs kanseri hücreleri kullanır. Ayrıntılı seçim süreci ve bu seçilen hücre hatları özellikleri daha önce 24 tarif edildi. </li><li> Dulbecco tadil edilmiş Eagle ortamı (DMEM) ve fetal sığır serumu (FBS), 10%, 90% oluşan ve temel olmayan amino asitler ile takviye edilmiş bir ortam içinde, 37 ° C&#39;de 10 cm kültür tabaklarında tüm hücreleri büyütün. % 5 CO2 atmosferinde ortam muhafaza edin. 5 nM dosetaksel de MCF-7 TXT hücreleri korumak. </li><li> canlı görüntüleme için lamelleri Tohum hücreleri. Gecede UV onları teşhir sonra% 70 alkol ile durulama ve 24 mm poli-L-lizin kaplı cam lamelleri sterilize edin. 35 mm&#39;lik bir 6-yuvalı kültür plakasına lamelleri yerleştirin. plHer iyi bir lamel ace. </li><li> 10 cm&#39;lik bir kültür kabı orta çıkarın ve hücreler PBS ile yıkayın. çanak hücreleri ayırmak için çanak tripsin-EDTA 0.5 mL ekleyin. Hücrelerin dekolmanı aşağıdaki tripsin nötralize etmek çanak kültür ortamı 5 ml ekleyin. </li><li> Birim hacim başına hücre sayısını belirlemek için bir hemasitometre ile hücre sayımı. </li><li> Her bir birim hacmi başına hücre sayısı göre yaklaşık olarak 10 5 hücre ekleyin. hafifçe sallayarak sonra, hücreler lamelleri eklemek için izin inkübatör geri tabak koydu. </li></ol></li><li> GFP etiketli α-tubulin ekspresyonu <ol><li> Mikrotübül dinamiklerinin tahlil üreticinin protokolüne göre, ticari GFP transdüksiyon kontrolü (ör BacMam) GFP-etiketli α-tubulin ile hücreleri nakletmek için. </li><li> 37 ° C hücre kültürü inkübatöründe 36 saat bir gözdeki hücrelerin com izin vermek için, kültürkomplet yapışma ve% 60 izdiham. </li><li> aşağıdaki denkleme göre, her bir oyuğa ilave GFP-etiketli α-tubulin uygun hacimde hesaplayın: <img alt="Denklem" src="/files/ftp_upload/55027/55027eq1.jpg" /> hücrelerin sayısı etiketleme sırasında bir gözdeki hücrelerin sağ tahmini toplam sayısıdır ve istenen PPC hücre başına parçacıkların sayısıdır. Not: MCF-7 hücreleri tohumlama 20 arzu edilen bir PPC 10 5 5 10 ila 2 x hücre sayısı iki gereken her bir lamel için gerekli hacmi dolayısıyla 40 ml. farklı hücreler için, hacim en iyi olmayabilir yukarıda verilen formüle göre hesaplanacaktır. Kesin hacmin GFP tubulin gerçek ifade seviyesine göre ayarlanmalıdır. </li><li> homojen bir çözelti sağlamak için ters çevirme ile GFP etiketli α-tübülin birkaç kez karıştırılır. vorteks etmeyin. NOT: kontrolü için, boş (kontrol) reaktif herhangi bir memeli genetik yoksunelemanları ve potansiyel bakulovirüs dolayımlı etkileri ve arka plan floresanı belirlenmesine yardımcı olmak için kullanılabilir. Olmayan hedefli GFP transdüksiyon kontrolü de tüm hücre yanacaktır, hangi kullanılabilir. </li><li> taze ortam ile kültür ortamı değiştirin. Her bir oyuğa GFP etiketli α-tubulin, yeni orta 2 mL 40 uL ekleyin. </li><li> kültür ortamı ile GFP etiketli α-tubulin tamamen karışmanın sağlanması için hafifçe plakayı sallayın. Kültür Plakaları kuluçka makinesine geri dönün ve GFP-tubulin ekspresyonunu sağlamak için 24 saat boyunca inkübe edilir. </li></ol></li><li> Dosetaksel ile muamele hücreleri Not: Bu deney mikrotübül dinamik istikrarsızlık üzerinde dosetaksel etkisini test etmektir. Bu durumda, hücreler, dosetaksel istenen konsantrasyona ile muamele edilir. <ol><li> Orta DMEM% 90 FBS ve% 10 oluşur ve temel olmayan amino asitler ile takviye hazırlayın. fenol kırmızısı flüoresan etkileyebilir çünkü fenol kırmızısı olmadan DMEM kullanınGFP-tubulin CE. (10 nM ila 10 uM arasında) doketaksel istenilen konsantrasyonu ekleyin. dosetaksel stok solüsyonları dimetil sülfoksitte (DMSO) içinde 10 mM&#39;dir. </li><li> Ön sıcak doku kültürü kuluçka makinesi içinde 37 ° C docetaksel içeren ortam. </li><li> docetaksel içeren ortam ile normal kültür ortamı yerine ve doku kültürü kuluçka makinesi içinde 30 dakika inkübe edilir. Her bir dosetaksel konsantrasyonu için oyuk başına en az 3 lamelleri olmalıdır. </li></ol></li></ol> 2. Canlı Görüntüleme Mikrotubul dinamik dengesizlik incelemek için <ol><li> Sistem kurma <ol><li> 37 ° C&#39;de ve% 5 CO2 de muhafaza edilen bir oda içinde deneyleri gerçekleştirmek. Bir 60X, ters floresan mikroskop ve bir CCD kamera ile 1.42 NA petrol objektif lens ile donatılmış bir mikroskobik sistemi ile zaman atlamalı tarafından floresan görüntü kazanır. </li><li> canlı görüntüleme için hücreler ayarlayın. Pre-sıcak numune tutucu hem de37 ° C&#39;ye kadar ortam. incelemek için lamel seçin. Bir numune tutucu üzerinde ilgi lamel monte ve aşama 1.3.1 hazırlanan docetaksel içeren ortam 1 ml ile inkübe edin. </li></ol></li><li> Mikrotübül dinamik istikrarsızlık Canlı görüntüleme <ol><li> görüntüleme için kullanılacak hücrelerin belirlenmesi. Bunlar, düz GFP-tubulin yüksek floresan yoğunluğunu, ve açık bir mikrotübül yapılarının sahip olmalıdır. </li><li> İlk incelemek için aşama 2.2.1 üzerinden tanımlanmıştır hücre grubundan bir ölçeri seçin. belirlenen hücrenin çevresel alana odaklanmak. maruz kalma süresi için program ve görüntü alımı sıklığını ayarlayın. Pozlama süresi genellikle 0,5 s gibi görüntüleri her iki s satın alınacak. </li><li> 1.33 den 2.2.3 adımları için ek bir 20 dakika bekleyin. Aşama 1.3.3 tarif edildiği gibi nedenle, tam 50 dakika dosetaksel ilave edildikten sonra (bu standardizasyon için) görüntüleme başlar. </li><li> mikrotübül d kaydedindinamiği çalışmaları 2 dakika süreyle, bir Fotoğrafa her 2 sn alarak. Toplamda, tespit hücre için 60 görüntü kazanır. </li><li> Bir sonraki tanımlanmış hücreye taşımak ve tekrar 2.2.4 için 2.2.2 adımları. 5 hücreleri görüntülü kadar aynı işlemi tekrarlayın. Adım 1.3.3 de açıklandığı gibi zaman, dosetaksel eklenmesinden sonra 70 dakika ulaşmadan önce bunu yapmak zorunludur. </li><li> Her tedavi koşulu için tekrarlayın 3 farklı lamelleri için 2.2.5 için 2.2.1 adımları. Toplamda, görüntü 15 hücreleri, yeterli veri mikrotübül dinamik istikrarsızlık ölçmek için hangi. </li></ol></li></ol> 3. Görüntü İşleme ve Veri Analizi <ol><li> Görüntülerin Dekonvolüsyon <ol><li> yüksek kalitede elde etmek için elde edilen görüntüleri Deconvolve. mikroskop sistemi ile donatılmış Dekonvolüsyonun programını açın. </li><li> &quot;Süreç&quot; tıklayın ve &quot;Deconvolve.&quot; &quot;Giriş&quot; penceresinde görüntü dosyasını sürükleyin ve tıklayın &quot;Do.&quot; görüntü dosyası deconvolved ve olacakdeconvolved dosya otomatik olarak yeni bir dosya olarak kaydedilir. </li></ol></li><li> Videoları Üretimi <ol><li> donanımlı yazılımla açmak için deconvolved görüntü dosyasını tıklatın. </li><li> &quot;Dosya&quot; tıklayın ve &quot;film olarak kaydedin.&quot; olarak &quot;AV&quot; ve &quot;Animasyon tarzı&quot; olarak &quot;Film formatı&quot; Seç &quot;İlet.&quot; &quot;100%&quot; ve &quot;Çerçeve oranı&quot; için &quot;Sıkıştırma kalitesi&quot; Set &quot;5 / saniye.&quot; </li><li> &quot;Zaman içinde canlandırın&quot; ve tıklayın kutusunu işaretleyin &quot;Do it.&quot; Video oluşacaktır. </li></ol></li><li> Mikrotübül kısalma ve büyüme hızının ölçülmesi <ol><li> ölçümü için kullanılacak mikrotübül belirleyin. her bir hücre için, sadece bir kaç mikrotübüller bunların yağ ya da büyüme açık ve tam olarak takip olması mümkün olur. </li><li> mikrotübül a başlangıç ​​uzunluğunu gösterir çerçeveyi belirlemek için videonun her karesini inceleyinnd en uzatılabilir veya kısaltılabilir mikrotübül ile çerçeve. İki mikrotübül ve iki kare arasında geçen süre arasındaki uzunluk değişimi ölçün. </li><li> zaman uzunluğu değişimi bölerek büyüme veya kısalma oranını hesaplamak (birim mm / s). Her bir tedavi durumu için, en az 10 hücrelerin takip ve en az 20 mikrotübülleri ölçer. </li><li> Her bir tedavi durumu için ortalama ve standart hata hesaplanır. Öğrenci testi kullanılarak koşulları arasındaki istatistiksel fark analiz eder. tablo ve / veya bir grafik veri rapor edin. </li></ol></li></ol>

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Yazarlar hiçbir rakip mali çıkarları olduğunu beyan ederim.

In vitro ve in vivo: mikrotübül Dinamik dengesizlik ölçmek için iki ana yöntem vardır. In vitro yöntemde, saflaştırılmış tubulin bilgisayar destekli zaman atlamalı diferansiyel girişim kontrast mikroskobu ile mikrotübül dinamik istikrarsızlık ölçmek için kullanılır. In vivo metodunda, floresan tubulin mikroenjeksiyon ya da GFP-tubulin ifade mikrotübüllere dahil edilir. Mikrotübül dinamikleri (büyüme ve kısalma) sonra fazlar arası hücrelerinin ...

<html> Meme kanseri ölümlerinin önde gelen nedenidir metastazı 1, 2 geçer. Birinci basamak metastatik meme kanseri, 2, 3, 4, 5, 6 tedavisinde rejimleri gibi dosetaksel ve paklitaksel gibi taksanlarm kendilerine hidrofobik şu anda kullanılmaktadır. Onlar mikrotübül dinamiklerini bozabilir mikrotübül hedefleme ajanları (MTAs) bir grubun parçasıdır. Ancak, küratif tedavi taksan kullanarak en büyük sorunlardan biri nüks 7 neden kanser hücrelerinde taksan direnci, geliştirilmesidir. İlaç direnci metastatik meme kanseri 7 olan hastalarda, tüm ölümlerin% 90&#39;dan fazla sorumludur. Mikrotübüller α- ve β-tübülin heterodimerlerinin polimerizasyonuyla oluşurclass = &quot;xref&quot;&gt; 8, 9. mikrotubul dinamiği kesin düzenlemesi, hücre polarizasyon, hücre döngüsü ilerlemesinin, hücre içi ulaşım ve hücre sinyalizasyon dahil olmak üzere birçok hücre fonksiyonları için önemlidir. Mikrotübüllerin ve dinamikleri bozulması hücre fonksiyonunu bozabilir ve hücre ölümü 10, 11 neden olur. Bu sistem bozukluğu neden bağlı olarak, MTA ilaçlar mikrotübül stabilize edici ajanlar (örneğin, taksanlar) ya da mikrotübül destabalizing maddeler (örneğin, vinka alkaloidler ve kolşisin yerinde bağlama maddeleri), 20, ya da sınıflandırılabilir. Mikrotübül kütlesi üzerindeki zıt etkilerine rağmen, yeterli dozda, hem sınıflar mikrotübül dinamiklerine 21 üzerindeki etkileri yoluyla kanser hücrelerini öldürür. Taksan yol açan mikrotübül mili 12 dengeleyici öncelikle işlevkromozomal hiza. mili montaj kontrol noktasında (SAC) sonraki sürekli aktivasyon mitoz hücreyi tutuklamalar. Uzamış mitotik tutuklama daha sonra apoptoz 13, 14 olur. Taksan monte tubulin 16 sadece mevcut olan β-tubulin 8, 15, ilgili taksan bağlanma sitesi üzerinden mikrotübüller ile etkileşime girer. Taksan direnç çok mekanizma 17 9 önerilmiştir. Bu mekanizmalar sayesinde ilaç akış proteinler ve taksan özgü direnci 5, 9, 18, 19 aşın hem genel hem de çoklu ilaç direncini kapsamaktadır. Örneğin, taksan dirençli kanser hücreleri, bazı β-küvet ekspresyonu ve fonksiyonunu değiştirmiş olabilirUlin 5, 9, 19, 20, 21, 22, 23 izotipleri. Mikrotübül Dinamik dengesizlik ölçmek için bir in vivo yöntem kullanarak, dirençli olmayan ebeveyn MCF-7 hücreleri CC 17 ile karşılaştırıldığında, dosetaksel dayanıklı MCF-7 TXT hücrelerin mikrotübül dinamiklerinin dosetaksel tedaviye duyarlı olduğunu göstermektedir. Daha iyi MTAs işlevini ve kanserli hücrelerde taksan direnci tam mekanizmasını anlamak için, mikrotübül dinamiklerini ölçmek için esastır. Burada, bu sayede, bir in vivo yöntem sunulmaktadır. Hücrelerinde GFP-etiketli tubulin sentezlenmesi ile birlikte canlı görüntüleme kullanarak, MCF-7 TXT ve MCF-7 hücreleri CC ve wi mikrotübül dinamiklerinin ölçebilirthout dosetaksel tedavisi. Sonuçlar bize taksan direncini aşmak için daha etkili ilaçlar tasarlamaya yardımcı olabilir.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Life Technologies, Invitrogen</td><td>11140-050</td><td></td></tr><tr><td>FBS</td><td>Gibco, Invitrogen</td><td>12483</td><td></td></tr><tr><td>Anti-Anti (100x)</td><td>Life Technologies, Invitrogen</td><td>15240-062</td><td></td></tr><tr><td>docetaxel</td><td>Sigma-Aldrich</td><td>01885-5mg-F</td><td></td></tr><tr><td>DMEM phenol red-free</td><td>Gibco, Invitrogen</td><td>21063</td><td></td></tr><tr><td>CellLight Reagent *BacMam 2.0* GFP-tubulin</td><td>ThermoFisher Scientific</td><td>C10613</td><td>Key reagent for expressing GFP tubulin in cells</td></tr><tr><td>CellLight Reagent *BacMam 2.0* GFP</td><td>ThermoFisher Scientific</td><td>B10383</td><td>Control</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich+B9:AA9</td><td>472301</td><td>for dissoving decetaxel</td></tr><tr><td>22-mm glass coveslip</td><td>Fisher Scientifics</td><td>12-545-101</td><td></td></tr><tr><td>6-well culture plate</td><td>Greiner Bio-One International</td><td>6 Well Celi Culture Plate</td><td></td></tr><tr><td>DeltaVision Microscopy Imaging Systems</td><td>GE Health</td><td></td><td>This system is equipped with weather station for controlling temperature and CO2. It also equipped with Worx Software for deconvolution and time lapse control.</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>ThermoFisher Scientific</td><td>25200056</td><td></td></tr><tr><td>Bright-Line Hemacytometer Set, Hausser Scientific</td><td>Hausser Scientific, Distributed by VWR</td><td>Supplier No.: 1492 VWR No.:15170-172</td><td></td></tr></tbody></table>

live imaging to study microtubule dynamic instability in taxane-resistant breast cancers

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane resistance in cancerous cells drastically reduces the effectiveness of the drugs' long-term usage. Accumulated evidence suggests that the mechanisms underlying taxane resistance include both general mechanisms, such as the development of multidrug resistance due to the overexpression of drug-efflux proteins, and taxane-specific mechanisms, such as those that involve microtubule dynamics. 
Because taxanes target cell microtubules, measuring microtubule dynamic instability is an important step in determining the mechanisms of taxane resistance and provides insight into how to overcome this resistance. In the experiment, an in vivo method was used to measure microtubule dynamic instability. GFP-tagged &#945;-tubulin was expressed and incorporated into microtubules in MCF-7 cells, allowing for the recording of the microtubule dynamics by time lapse using a sensitive camera. The results showed that, as opposed to the non-resistant parental MCF-7CC cells, the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitive to docetaxel treatment, which causes the resistance to docetaxel-induced mitotic arrest and apoptosis. This paper will outline this in vivo method of measuring microtubule dynamic instability.

Burada sunulan protokolü kullanarak, (MCF-7 TXT) meme kanseri hücrelerinde (MCF-7 CC), normal ve dosetaksel dayanıklı mikrotübül dinamiklerine dosetaksel etkilerini inceledi. Görüntü iki takım MCF-7 CC ve MCF-7 TXT hücreleri (Şekil 1A) mikrotübül büyüme ve katı üzerine dosetaksel (0.5 uM) etkilerini göstermektedir. Ayrıca, iki hücre hatları içi...

Watch this Scientific Journal Video about Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers at JoVE.com

Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells.

Canlı Görüntüleme taksan dirençli Meme Kanserlerinde Mikrotubul dinamik dengesizlik Eğitim için

Taxanes such as docetaxel belong to a group of microtubule-targeting agents (MTAs) that are commonly relied upon to treat cancer. However, taxane ...

live-imaging-to-study-microtubule-dynamic-instability-taxane

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7.5K Görüntüleme.  University of Alberta. In this paper, we report a protocol describing an in vivo method to measure microtubule dynamic instability in docetaxel-resistant breast cancer cells (MCF-7TXT). In this method, a deconvolution microscopy imaging system is used to detect the expression of GFP-tubulin in target cells. 

Video: Live Imaging to Study Microtubule Dynamic Instability in Taxane-resistant Breast Cancers

Live-imaging of Breast Epithelial Cell Migration After the Transient Depletion of TIP60

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the beginning and end of an experiment using a phase-contrast microscope, and the migration abilities of cells are evaluated by the closure of wounds. However, cell movement is a dynamic phenomenon, and a conventional method does not allow for tracking single-cell movement. To improve current wound-healing assays, we use live-cell imaging techniques to monitor cell migration in real time. This method allows us to determine the cell migration rate based on a cell tracking system and provides a clearer distinction between cell migration and cell proliferation. Here, we demonstrate the use of live-cell imaging in wound-healing assays to study the different migration abilities of breast epithelial cells influenced by the presence of TIP60. As cell motility is highly dynamic, our method provides more insights into the processes of wound healing than a snapshot of wound closure taken with the traditional imaging techniques used for wound-healing assays.

Here, we present the real-time monitoring of cell migration in a wound-healing assay using TIP60-depleted MCF10A breast epithelial cells. The implementation of live-cell imaging techniques in our protocol allows us to analyze and visualize single-cell movement in real time and across time.

Meme epitel hücre göç TIP60 geçici tükenmesi sonra canlı görüntüleme

The wound-healing assay is efficient and one of the most economical ways to study cell migration in vitro. Conventionally, images are taken at the ...

Kanser Araştırmaları

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Biyoparlaklık ve Micro CT Görüntüleme Tümör Metastazlari ve osteolitik Lezyonlar İzleme

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Tıp

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Uzun vadeli canlı hücre kader Paklitaksel karşılık olarak değerlendirmek için Imaging hücreli

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Meme Kanseri Fare Modelleri Tümör Mikroçevre İlaç Yanıtlarının Görüntüleme Canlı

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Birincil Preneoplastic Meme Epitel Hücrelerinin Time-lapse görüntüleme Meme Kanseri Genetik Fare Modelleri türetilen

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques enable the observation of live cellular behavior inside the organ but can be challenging in some types of cancer due to organ and tumor accessibility such as intestine. Direct observation of intestinal tumors has not been previously reported. A surgical procedure described here allows direct observation of myeloid cell dynamics within the intestinal tumors in live mice by using transgenic fluorescent reporter mice and injectable tracers or antibodies. For this purpose, a four-color, multi-region, micro-lensed spinning disk confocal microscope that allows long-term continuous imaging with rapid image acquisition has been used. ApcMin/+ mice that develop multiple adenomas in the small intestine are crossed with c-fms-EGFP mice to visualize myeloid cells and with ACTB-ECFP mice to visualize intestinal epithelial cells of the crypts. Procedures for labeling different tumor components, such as blood vessels and neutrophils, and the procedure for positioning the tumor for imaging through the serosal surface are also described. Time-lapse movies compiled from several hours of imaging allow the analysis of myeloid cell behavior in situ in the intestinal microenvironment.

Real-time Imaging of Myeloid Cells Dynamics in ApcMin/+ Intestinal Tumors by Spinning Disk Confocal Microscopy

By using transgenic reporter mice and injectable fluorescent labels, long-term intravital spinning disk confocal microscopy enables direct visualization of myeloid cell behavior into intestinal adenoma in the ApcMin/+ colorectal cancer model.

Miyeloid hücrelerin Dynamics Gerçek zamanlı Görüntüleme<em&gt; Süm<sup&gt; Min / +</sup</em&gt; Bağırsak Tümörleri Disk Eşodaklı Mikroskopi Spinning tarafından

Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

İmmünoloji ve Enfeksiyon

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and networks often contain subsets of microtubule arrays that differ in their dynamic properties. For example, in dividing cells, stable bundles of crosslinked microtubules coexist in close proximity to dynamic non-crosslinked microtubules. TIRF-microscopy-based in vitro reconstitution studies enable the simultaneous visualization of the dynamics of these different microtubule arrays. In this assay, an imaging chamber is assembled with surface-immobilized microtubules, which are either present as single filaments or organized into crosslinked&#160;bundles. Introduction of tubulin, nucleotides, and protein regulators allows direct visualization of associated proteins and of dynamic properties of single and crosslinked microtubules. Furthermore, changes that occur as dynamic single microtubules organize into bundles can be monitored in real-time. The method described here allows for a systematic evaluation of the activity and localization of individual proteins, as well as synergistic effects of protein regulators on two different microtubule subsets under identical experimental conditions, thereby providing mechanistic insights that are inaccessible by other methods.

Simultaneous Visualization of the Dynamics of Crosslinked and Single Microtubules In Vitro by TIRF Microscopy

Here, a TIRF microscopy-based in vitro reconstitution assay is presented to simultaneously quantify and compare the dynamics of two microtubule populations. A method is described to simultaneously view the collective activity of multiple microtubule-associated proteins on crosslinked microtubule bundles and single microtubules.

Çapraz Bağlı ve Tek Mikrotübüllerin Dinamiğinin In Vitro TIRF Mikroskobu ile Eşzamanlı Görselleştirilmesi

Microtubules are polymers of &#945;&#946;-tubulin heterodimers that organize into distinct structures in cells. Microtubule-based architectures and ...

Biyokimya

Through the Looking Glass: Time-lapse Microscopy and Longitudinal Tracking of Single Cells to Study Anti-cancer Therapeutics

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing factor in the overall outcome. Immunoblotting, flow cytometry and fixed cell experiments are often used to study how cells respond to anti-cancer drugs. These methods are important, but they have several shortcomings. Variability in drug responses between cancer and normal cells, and between cells of different cancer origin, and transient and rare responses are difficult to understand using population averaging assays and without being able to directly track and analyze them longitudinally. The microscope is particularly well suited to image live cells. Advancements in technology enable us to routinely image cells at a resolution that enables not only cell tracking, but also the observation of a variety of cellular responses. We describe an approach in detail that allows for the continuous time-lapse imaging of cells during the drug response for essentially as long as desired, typically up to 96 hr. Using variations of the approach, cells can be monitored for weeks. With the employment of genetically encoded fluorescent biosensors numerous processes, pathways and responses can be followed. We show examples that include tracking and quantification of cell growth and cell cycle progression, chromosome dynamics, DNA damage, and cell death. We also discuss variations of the technique and its flexibility, and highlight some common pitfalls.

Here, we describe a method of long-term time-lapse microscopy to longitudinally track single cells in response to anti-cancer therapeutics.

Looking Glass sayesinde: Time-lapse Mikroskopi ve Tek Hücre Boyuna izleme Anti-kanser Therapeutics Eğitim için

The response of single cells to anti-cancer drugs contributes significantly in determining the population response, and therefore is a major contributing ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Tümör mikroçevresinde Gerçek zamanlı Multisellüler Dynamics Genişletilmiş Time-lapse intravital Görüntüleme

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Yüksek çözünürlüklü görüntüleme ve Bireysel Astral Mikrotubul Dynamics Analizi Budding Maya

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...