Avustralya Nükleer Bilimi ve Mühendisliği Enstitüsü destek kabul edilmektedir. TCK AINSE ödül layık görüldü. Epigenomic Tıp Laboratuarı, Ulusal Sağlık ve Tıbbi Araştırma Konseyi (566.559) Avustralya tarafından desteklenmektedir. İK, Avustralya, mezuniyet sonrası ve BakerIDI parlak kıvılcım ödül tarafından desteklenmektedir. Bu çalışma, Biyomedikal Görüntüleme Geliştirme Ltd (CRC-BID), kurulan ve Avustralya Hükümeti Ortak Araştırma Merkezleri programı kapsamında desteklenen CRC tarafından finanse edilmektedir. CO Avustralyalı bir mezuniyet sonrası ödül ve CRC-BID ek burs almıştır.

1. Hücre Kültürü ve Deneysel Set-up <ol><li> İnsan keratinositler keratinosit-SFM 15 mL (K-SFM) içeren 75 cm 2 doku kültürü şişeleri mono tabakaları olarak korunur, L-glutamin (2 mM), epidermal büyüme faktörü (5 ng ile desteklenmiş orta (Gibco, serum serbest orta ) / ml), sığır hipofiz ekstresi (40 mg / ml) ve 20 mg / ml gentamisin. Hücreler 37 ° C nemlendirilmiş bir% 5 CO 2 ortamında yetiştirilen </li><li> Hücreler, 5 ml K-SFM orta içeren 12 x 25 cm 2 doku kültürü şişeler içine numaralı seribaşı . </li></ol> Tek hücre süspansiyonları trypsinization tarafından hazırlanır. Hücreler, fosfat tamponlu tuzlu su ile yıkanır ve 5-10 dakika için% 0.05 tripsin / EDTA çözeltisi ile inkübe edilir. Hücreler haline başladığınızda yuvarlak ve müstakil ~% 30,% 10 fetal sığır serumu içeren Dulbecco&#39;nun modifiye kartal orta 3 cilt tripsin nötralize etmek için eklenir. Hücreleri (20 kez) yukarı ve aşağı pipetleme ayrılır. Hücreler hemasitometre kullanarak sayılır. Uygun hücre sayıları iki katına zaman hücre hattı (insan FEP-1811 keratinositler için yaklaşık 20 saat) göre seribaşı. Amacı, deney günü ~% 90 confluency (şişesi ortalama ~ 10 6 hücre) elde etmektir . 12 şişe oluşan bir deney yaklaşık dört saat içinde tamamlanmalıdır tek bir clonogenic assay (altı ışınlanmamış kontrolü ve altı ışınlanmış şişeler) için en uygunudur. 2. Tedavi ve ışınlama <ol><li> Ilgili bir radyasyon değiştirerek bileşik uygun bir zaman için hücreleri tedavi ve hücreleri iyonize radyasyon γ-radyasyon veya X-ışınları ya maruz. </li></ol> Genellikle altı şişeler, kaplama verimlilik (işlenmemiş) ve ilaç denetimlerin sadece olarak hizmet vermektedir. Diğer altı şişeler ışınlanmış. 1 saat suda eriyen bir doğal antioksidan formülasyonu, bu örnekte insan keratinositler Cinnulin PF çeşitli konsantrasyonlarda (20 mg / ml aşağıda gösterilmiştir temsilcisi veri; Dürüstlük Nutraceuticals Uluslararası, Spring Hill, TN, ABD CPF) ile tedavi edilir 37 ° C Hücreler 137 Cs kaynak (Nordion Uluslararası, ON, Kanada; 1.6 Gy / dk Gammacell 1000 Elite ışınlama) kullanılarak 4 Gy ışınlanmış. 3. Kaplama <ol><li> Daha önce açıklandığı gibi, tedavisi sonrasında, tek hücre süspansiyonları elde edilir. </li><li> Her bir numune hücrelerinin sayısını dikkatli bir şekilde hemasitometre kullanarak, uygun hücre sayıları (beş 15 mm yemekleri her çoğaltır) petri kutularının içine numaralı seribaşı sayılır ve seyreltilir. Plaka başına tohum hücrelerinin sayısını karar verirken kaplama verimliliği ve / veya fraksiyon hayatta tahmin edilmelidir. 150 koloniler - 20 arasında bir dizi amacı gerçekleştirmek için. </li></ol> Petri kutularına nemlendirilmiş bir plastik klonlama kutusu olarak düzenlenmiş ve koloni oluşumu için 37 ° C,% 5 CO 2 ortamında inkübe . Koloni oluşumu için inkübasyon süresi 1-3 hafta farklı hücre hatları için değişir, o zaman en az altı hücre bölünmeleri eşdeğer olması gerektiğini kabul edilir. Bu örnekte, insan keratinositler için kontrol yemekleri sekiz gün, 50 ya da daha fazla hücreden oluşan yeterince büyük klonlar formu gerektirir. 4. Tespit ve Boyama Kolonileri Davlumbaz aşağıdaki adımları tamamlayın. <ol><li> Aspirasyon ile hafifçe plakaların her medya çıkarın. </li><li> Her plaka 5 ml% 0.9 &#39;luk tuzlu su ile yıkayın. </li><li> 15-30 dakika için 5 ml% 10&#39;luk nötral tamponlu formalin ile koloniler sabitleyin. </li><li> 30-60 dakika dH 2 O 5 ml% 0,01 (w / v) kristal viyole ile Leke. </li><li> DH 2 O ile aşırı kristal viyole yıkayın ve yemekleri kurumasını bekleyin. </li></ol> 5. Koloni Sayım Stereomikroskopta <ol><li> 50 den fazla bireysel hücre içeren Kolonileri stereomikroskopta kullanarak sayılır. </li></ol> Dijital görüntüleme ve görüntüleme yazılımı kullanarak sayma <ol><li> Kolonilerin Dijital görüntüler bir kamera kullanarak ya da cihaz tarama elde edilir </li><li> Kolonileri aşağıda açıklandığı gibi görüntü analiz yazılımı paketleri kullanarak sayılır. </li></ol> ImageJ (Fiji Sürüm 1.44a) kullanarak hücre sayımı <ol><li> Fiji görüntü dosyasını açın, Dosya -&gt; Aç. </li><li> &gt; Ayarlayın ...-&gt; Eşik görüntü 8-bit formatına dönüştürebilirsiniz Gerekirse, Görüntü gitmek. </li><li> Böylece sadece koloniler tespit non-spesifik arka plan seviyesini düşürmek için eşik ayarlayın. </li><li> Aşağıdaki kullanarak kolonilerin sayısı: Proses -&gt; Binary -&gt; maxima bul. </li></ol> Bu görüntü formatı için, gürültü toleransı 0 olarak ayarlanır. O ışık arka plan seçeneği işaretli olduğundan emin olun vetespit maxima önizleme tüm hücre kolonileri doğru kayıtlı olup olmadığını kontrol edin.

<ol>
	<li>Puck, T. T., Marcus, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+x-rays+on+mammalian+cells.">Action of x-rays on mammalian cells.</a> J Exp Med. 103, 653-666 (1956).</li><li>Hurlin, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+human+papillomavirus+type+18-immortalized+human+keratinocytes+to+a+malignant+phenotype.">Progression of human papillomavirus type 18-immortalized human keratinocytes to a malignant phenotype.</a> Proc Natl Acad Sci U S A. 88, 570-574 (1991).</li></ol>

Bu örnekte, 100 mcg / ml CPF insan FEP-1811 keratinositler çeşitli konsantrasyonlarda ile tedavi edildi; veriler 37 1 saat için 20 mg / ml CPF ° C için gösterilen Ardından tedavi hücreleri 137 Cs kaynak (Nordion Uluslararası, ON, Kanada; 1.6 Gy / dk Gammacell 1000 Elite ışınlama) kullanılarak 4 Gy ışınlanmış. Tedavi edilmemiş ve ilaç tedavileri 100 hücre kontrolü için her bir petri kaplama ve çanak başına 1000 hücreleri ışınlanmış örnekler için kaplanmıştır. Yukarıdaki t...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Keratinocyte-serum free medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>17005042</td>
<td>Supplemented with 2mM L-glutamine, 20&#x03BC;g/ml gentamicin, epidermal growth factor, bovine pituitary extract.
</td>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15400-054</td>
<td>0.05% trypsin / EDTA to detach adherent cells; 10 x stock diluted in PBS w/o Ca2+/Mg2+.</td>
</tr>
<tr>
<td>Dulbecco&#x2019;s modified essential medium</td>
<td>Growth medium</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>Supplemented with 10% FBS and 20&mu;g/ml gentamicin; used to neutralise trypsin/EDTA.</td>
</tr>
<tr>
<td>0.09% Saline</td>
<td>Solution</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td> Used to wash colonies.</td>
</tr>
<tr>
<td>10% Neutral buffered formalin solution</td>
<td>Solution</td>
<td>Sigma-Aldrich</td>
<td>HT501128</td>
<td>~4% formaldehyde; used to fix colonies.</td>
</tr>
<tr>
<td>Crystal violet</td>
<td>Powder</td>
<td>SPI-Chem</td>
<td>02577-MB</td>
<td>0.01% crystal violet solution, prepared in dH2O, used to stain colonies.</td>
</tr>
<tr>
<td>Gammacell 1000 elite irradiator</td>
<td>&nbsp;</td>
<td>Nordion International Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Petri dishes</td>
<td>60 x 15 mm</td>
<td>Falcon</td>
<td>353002</td>
<td>&nbsp;</td>
<tr>
<td>Haemocytometer</td>
<td>&nbsp;</td>
<td>Hawksley; Medical and Laboratory Equipment</td>
<td>AC1000</td>
<td>&nbsp;</td>
<tr>
<td>Cloning box</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Plastic box with holes punched at opposite sides of lid; for storing petri dishes during prolonged incubation for colony formation.</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Type 102</td>
<td>Nikon</td>
<td>&nbsp;</td>
<td>Used to count colonies consisting of &gt;50 cells.</td>
</tr>		</tbody>
		</table>
		</div>

clonogenic assay: adherent cells

Clonogenic (ya da koloni oluşturan) testinin, 50 yılı aşkın kurulmuştur; 1956 1 tekniğini anlatan orijinal kağıt yayımlandı. Yöntemini belgeleyen dışında, ilk dönüm noktası çalışma kültürü 1 X-ray ışınlanmış memeli (HeLa) hücreleri için ilk radyasyon doz yanıt eğrisi oluşturulur. Kontrolü tedavi edilmezse hücreleri ve bu tür maruz kalma gibi değişik işlemlerden geçirilmiş hücreleri arasında; Temelde, clonogenic testi üreme canlılığı farklılıkları (50 veya daha fazla hücre bir koloni oluşturmak için tek bir hücre yani döl hücreleri kapasitesi) bir değerlendirme sağlar radyasyon iyonize, çeşitli kimyasal bileşikler (örneğin sitotoksik ilaçlar) ya da diğer durumlarda genetik manipülasyon. Tahlil radyasyon biyolojisi en yaygın kabul gören bir tekniği haline gelmiştir ve farklı hücre hatları radyasyon duyarlılığı değerlendirmek için yaygın olarak kullanılır olmuştur. Ayrıca, clonogenic testi yaygın radyasyon değiştirerek bileşiklerin etkinliğinin izlenmesi ve koloni oluşturan farklı hücre hatları yeteneği, sitotoksik ajanlar ve diğer anti-kanser tedavileri etkileri belirlemek için kullanılır. Yapışık hücrelere hatları ile tipik bir clonogenic hayatta kalma deney koloniler tespit ve boyama üç farklı bileşenleri, 1), doku kültürü şişeler, hücre monolayer tedavisi, 2) tek bir hücre süspansiyonlarının hazırlanması ve uygun bir hücre sayısı petri kaplarına ve 3 kaplama) içerir. ilgili bir kuluçka dönemi sonrası, hangi hücre hattı bağlı olarak, 1-3 hafta arasında olabilir. İşte biz, bir ölümsüzleştirdi insan keratinosit hücre hattı kullanımı (FEP-1811) 2 ile yapışık hücre hatları ile clonogenic tahlil yapmak için genel prosedürü göstermektedir . Ayrıca, bizim hedeflerimize hücreleri radyasyona maruz kaldıktan sonra kaplama verimliliği ve hayatta kalma fraksiyonları hesaplama da dahil olmak üzere clonogenic testlerinin ortak özelliklerini tanımlamak ve doğal bir antioksidan formülasyonu kullanımı ile radyasyon cevap modifikasyonu örneklemek için.

Watch this Scientific Journal Video about Clonogenic Assay: Adherent Cells at JoVE.com

Clonogenic Assay: Adherent Cells

50 yıldan fazla üreme canlılığını değerlendirmek için clonogenic testin uygulanabilirliği için kurulmuştur. Burada yapışık hücrelere clonogenic tahlil yapmak için genel prosedür göstermektedir.

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. ...

Research

JoVE Journal

Biology

47.8K Görüntüleme.  The Alfred Medical Research and Education Precinct. 50 yıldan fazla üreme canlılığını değerlendirmek için clonogenic testin uygulanabilirliği için kurulmuştur. Burada yapışık hücrelere clonogenic tahlil yapmak için genel prosedür göstermektedir.

Video: Clonogenic Assay: Adherent Cells

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

İmalat ve Operasyon yapışkan Hücresel Kültürler Oksijen Ekle

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...

Biyoloji

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Yapışkan ve Sigara yapışık Memeli Hücre yerinde Subselüler Fraksiyonu

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

Tümör Hücrelerinin vaskülojenik Faaliyet değerlendirin bir Matrigel Tabanlı Tüp Oluşumu Testi

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Birincil Fare trofoblast hücreleri ve trofoblast invazyonu Assay İzolasyonu

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR muhabiri oluşturur ve bir microRNA kullanarak LightSwitch Lusiferaz Test Sistemi ile İnsan microRNA belirlenmesi Hedefler yapışık hücrelere Mimik

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

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 ...

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Gap Kavşak Sınavı Fonksiyonel Testi; İndiyum-Kalay Oksit Yapışık hücrelerin elektroporasyonu

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Dinamik Hücresel Protrüzyonlarda Protein Konsantrasyonunun Yazılım Destekli Takibi için Grafiksel Bir Kullanıcı Arayüzü

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

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

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 106 cell morphological measurements.

Lens-free video microscopy enables us to monitor cell cultures directly inside the incubator. Here we describe the full protocol used to acquire and analyze a 2.7 day long acquisition of cultured HeLa cells, leading to a dataset of 2.2 x 106 measurements of individual cell morphology and 10584 cell cycle tracks.

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 ...