Bu laboratuvarda glokom çalışmada Ulusal Glokom Araştırma, Amerikan Sağlık Yardımcısı Vakfı tarafından desteklenmektedir.

<ol><li> Cam pipetler, pipet çektirmenin kullanarak hazırlayın, 1 ml şırınga bağlamak ve parafilm ile bağlantı mühür. 2 ml solüsyon, pipet ucu içine çekiniz ve enjeksiyon için hazır olsun. </li><li> Ketamin (80mg/kg) ve xylazine (8mg/kg) (2:1 hacim oranı)-periton içi enjeksiyon yoluyla sıçan uyuşturan. </li><li> Sıçan gözleri intravitreal enjeksiyon öncesi lokal anestezik kremler olarak% 0.5 alcaine bir damla uygulayın. </li><li> Sıçan yerleştirin ve gözün üstün burun bölgesi maruz. </li><li> Ponksiyon par plana düzeyde üstün burun sklera 30 iğne kullanın. Oküler kas ve damarların dokunmaktan kaçının. </li><li> Vitreus küçük bir miktar arka kamara ponksiyonu delikten kurtulmak için göz topu hafif basınç verin. </li><li> Vitreus vücuda sklerasına ponksiyon delikten cam pipet ucu ile 45 derecelik bir açıyla yerleştirin. Arka odasına 2 ml solüsyon enjekte edilir ve birkaç saniye boyunca bir yerde saklayın, daha sonra iğne yavaşça çıkarın. Lens ve göz içi damarları dokunmaktan kaçının. </li><li> Intravitreal enjeksiyondan sonra, oftalmik Tobrex merhem sıçan göz enfeksiyonu önlemek için de geçerlidir. </li></ol>

<ol>
	<li>Chiu, K., Lam, T. T., Ying Ku, W. W., Caprioli, J., Kwong Kwong, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calpain+and+N-methyl-d-aspartate+(NMDA)-induced+excitotoxicity+in+rat+retinas.">Calpain and N-methyl-d-aspartate (NMDA)-induced excitotoxicity in rat retinas.</a> Brain Research. 1046, 207-215 (2005).</li><li>Fisher, D., Pavlidis, M., Thanos, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cataractogenic+lens+injury+prevents+traumatic+ganglion+cell+death+and+promotes+axonal+regeneration+both+in+vivo+and+in+culture.">Cataractogenic lens injury prevents traumatic ganglion cell death and promotes axonal regeneration both in vivo and in culture.</a> Investigative Ophthalmology &amp; Visual Science. 41, 3943-3954 (2000).</li><li>Ji, J. -. Z., Elyaman, W., Yip, H. K., Lee, V. W. H., Yick, L. -. W., Hugon, J., So, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNTF+promotes+survival+of+retinal+ganglion+cells+after+induction+of+ocular+hypertension+in+rats:+the+possible+involvement+of+STAT3+pathway.">CNTF promotes survival of retinal ganglion cells after induction of ocular hypertension in rats: the possible involvement of STAT3 pathway.</a> European Journal of Neuroscience. 19, 265-272 (2004).</li><li>Lam, T. T., Abler, A. S., Kwong, J. M. K., Tso, M. O. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-Methyl-D-Aspartate+(NMDA)-Induced+Apoptosis+in+Rat+Retina.">N-Methyl-D-Aspartate (NMDA)-Induced Apoptosis in Rat Retina.</a> Investigative Ophthalmology &amp; Visual Science. 40, 2391-2397 (1999).</li></ol>

Vizyon ve Oftalmoloji Tablolar Hong Kong Üniversitesi ve Araştırma Derneği, Eğitim ve Araştırma Canlı Hayvan Kullanımı (CULATR) Komitesi tarafından onaylanan araştırma hayvan kullanımı için protokole göre hayvanların ele alındı Oftalmik ve Vizyon Araştırma hayvanların kullanın.

İyi intravitreal enjeksiyon doğrudan göz içi tedavi yanı sıra hayvan modeli oluşturmak için yararlıdır. İlk olarak, enjeksiyon bölgesinde retina yapısını bozmayacak. İkincisi, kanama, enfeksiyon riskini azaltmak için kaçınılmalıdır. Üçüncü olarak, objektif, travmatik katarakt önlemek için bakir olmalıdır. En önemlisi, normal göz yapısı kesinti azaltmak için.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>
		<tbody>
		
<tr>
<td>Operating Microscope </td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>OME</td>
<td>&nbsp;</td>
<tr>
<td>Glass Pipettes </td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pipette Puller </td>
<td>&nbsp;</td>
<td>David Kopf Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1ml syringes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>single use</td>
</tr>
<tr>
<td>30G needles</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>single use</td>
</tr>
<tr>
<td>Ketamine</td>
<td>&nbsp;</td>
<td>Aldasan</td>
<td>&nbsp;</td>
<td>anesthetic</td>
</tr>
<tr>
<td>Xylazine</td>
<td>&nbsp;</td>
<td>Aldasan</td>
<td>&nbsp;</td>
<td>anesthetic</td>
</tr>
<tr>
<td>Alcaine</td>
<td>&nbsp;</td>
<td>Alcon-Couvreur</td>
<td>&nbsp;</td>
<td>0.5% proparacaine hydrochloride</td>
</tr>
<tr>
<td>Tobrex</td>
<td>&nbsp;</td>
<td>Alcon-Couvreur</td>
<td>&nbsp;</td>
<td>ointment, 3% tobramyxin.</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

intravitreous injection for establishing ocular diseases model

Intravitreal enjeksiyon görsel bilimler araştırma yaygın olarak kullanılan bir tekniktir. Göz hastalıkları ile veya doğrudan uygulama lokal tedavi olarak hayvan modellerinde kurmak için kullanılabilir. Bu video intravitreal enjeksiyon işlemi, basit ve ucuz araçların nasıl kullanılacağını tanıtır. Yerine, bir hemilton şırınga, 1 ml şırınga kullanın kullanılır. Uygun enjeksiyon iğneleri, mükemmel ipuçları ile cam pipetler kullanarak ve nasıl kolayca sıkıca cam pipet ile birlikte şırınga iğnesi bağlamak için ne yapmak için pratik ipuçları verilmektedir. 1 enjeksiyon yerinde retinanın yapısını bozmayacak); 2) kanama, enfeksiyon riskini azaltmak için kaçınılmalıdır; 3) travmatik katarakt lens önlemek için bakir olmalıdır: iyi bir intravitreal enjeksiyon yapmak için, dikkat edilmesi gereken üç yönü vardır. Özetle, en önemli nokta, normal göz yapısı kesinti azaltmaktır. Retinanın kesintiye uğramaması için, sıçan göz üstün burun bölgesi seçildi. Ayrıca, sıçan göz limbal bölgede yaklaşık 1.5 mm idi par düzlemsel, iğne ponksiyon noktası oldu. Vitreus küçük bir miktar hafifçe enjeksiyon öncesi göz içi basıncı azaltmak için ponksiyon delikten dışarı itilir. 45 ° enjeksiyon açısına sahip, böylece lentiküler faktörler ilgili komplikasyonlar ve etkisi kaçınarak, sıçan göz travmatik katarakt neden daha az olasıdır. Bu operasyonda, konjonktiva ve oküler kas hiçbir kesim, hiçbir kanama vardı. Hızlı ve küçük bir yaralanma ile başarılı bir intravitreal enjeksiyon dakika içinde yapılabilir. Enjeksiyon bu özel protokolde belirtilen değerini intravitreal enjeksiyon için özeldir. Ancak, burada sunulan yöntemler ve malzemeler aynı zamanda beyin, omurilik ya da diğer organlarda küçük memeliler, ilaç dağıtım, diğer enjeksiyon işlemleri için kullanılan olabilir.

Watch this Scientific Journal Video about Intravitreous Injection for Establishing Ocular Diseases Model at JoVE.com

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreal enjeksiyon, göz hastalıkları veya lokal tedavi doğrudan uygulama olarak görsel bilimler araştırma yaygın olarak kullanılan bir tekniktir. Bu video cam pipet yardımıyla 1 ml şırınga kullanarak intravitreal enjeksiyon için bir protokol gösterdi. Masif kanama ve lens hasarı önlemek konusunda yararlı ipuçları verilmiştir.

Oküler Hastalıkları Model kurulması için intravitreal enjeksiyon

Intravitreous injection is a widely used technique in visual sciences research. It can be used to establish animal models with ocular diseases or as ...

intravitreous-injection-for-establishing-ocular-diseases-model

Research

JoVE Journal

Biology

36.6K Görüntüleme.  The University of Hong Kong - HKU. Intravitreal enjeksiyon, göz hastalıkları veya lokal tedavi doğrudan uygulama olarak görsel bilimler araştırma yaygın olarak kullanılan bir tekniktir. Bu video cam pipet yardımıyla 1 ml şırınga kullanarak intravitreal enjeksiyon için bir protokol gösterdi. Masif kanama ve lens hasarı önlemek konusunda yararlı ipuçları verilmiştir.

Video: Intravitreous Injection for Establishing Ocular Diseases Model

Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Lazer Kaynaklı SD Sıçanlarda Kronik Oküler Hipertansiyon Modeli

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Biyoloji

Interview: Protein Folding and Studies of Neurodegenerative Diseases

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the protein folding is at the core of the modern biology. In addition to their traditional biochemical functions, proteins can mediate transfer of biological information and therefore can be considered a genetic material. This recently discovered function of proteins has important implications for studies of human disorders. Dr. Lindquist also describes current experimental approaches to investigate the mechanism of neurodegenerative diseases based on genetic studies in model organisms.

Röportaj: Protein Katlama ve Nörodejeneratif Hastalıklar Çalışmaları

In this interview, Dr. Lindquist describes relationships between protein folding, prion diseases and neurodegenerative disorders. The problem of the ...

Zebrafish Brain Ventricle Injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles. We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.

Zebra balığı Beyin Ventrikül Enjeksiyon

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

Oküler yüzdeki kütle spektrometresi esaslı proteomik analiz için numune hazırlama

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

EYFP etiketli CENP-A (EYFP-CENP-A) Ubiquitylation tanımlamak için Kütle Spektrometresi Analizi

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their detrimental effects on the human life quality and the healthcare system, the majority of human neurodegenerative disorders still remain incurable and non-preventable. Therefore, the development of novel therapeutic interventions against such maladies is becoming a pressing urgency. Age-associated deterioration of neuronal circuits and function is evolutionarily conserved in organisms as diverse as the lowly worm Caenorhabditis elegans and humans, signifying similarities in the underlying cellular and molecular mechanisms. C. elegans is a highly malleable genetic model, which offers a well-characterized nervous system, body transparency and a diverse repertoire of genetic and imaging techniques to assess neuronal activity and quality control during ageing. Here, we introduce and describe methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis (e.g., deg-3(d) and mec-4(d)) and protein aggregate (e.g., &#945;-syunclein and poly-glutamate)-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-related neuronal breakdown. A combination of these animal neurodegeneration models, together with genetic and pharmacological screens for cell death modulators will lead to an unprecedented understanding of age-related breakdown of neuronal function and will provide critical insights with broad relevance to human health and quality of life.

Modeling Age-Associated Neurodegenerative Diseases in Caenorhabditis elegans

Here, we introduce and describe widely accessible methodologies utilizing some versatile nematode models, including hyperactivated ion channel-induced necrosis and protein aggregate-induced neurotoxicity, to monitor and dissect the cellular and molecular underpinnings of age-associated neurodegenerative diseases.

Caenorhabditis elegans Yaş İlişkili Nörodejeneratif Hastalıkların Modellendirilmesi

Battling human neurodegenerative pathologies and managing their pervasive socioeconomic impact is becoming a global priority. Notwithstanding their ...

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6&#8210;8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC)&#160;transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

We show the automation of human induced pluripotent stem cell (hiPSC) cultures and neuronal differentiations compatible with automated imaging and analysis.

Entegre Canlı Hücre İzleme ile İnsan Kaynaklı Pluripotent Kök Hücre Kaynaklı Kortikal ve Dopaminerjik Nöronların Otomatik Üretimi

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Oküler Mikrovasküler Anjiyogenezin Ex Vivo Koroid Filizlenme Satomu

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Birincil Fare Retina Pigment Epitel Hücrelerinin Verimli Diseksiyonu ve Kültürü

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

UV Radyasyonu ve Kimyasallarının Primer ve Ölümsüzleştirilmiş İnsan Kornea Epitel Hücreleri Üzerindeki Toksisitesinin Belirlenmesi

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...