이 실험실에서 녹내장의 연구는 미국의 건강 보조 재단 국립 녹내장 연구에 의해 지원됩니다.

<ol><li> pipettes의 풀러를 사용하여 유리 pipettes 준비, 1ml 주사기에 연결하고 parafilm와 연결을 밀봉. pipettes 끝에 솔루션 2 ML을 철회하고 사출을위한 준비. </li><li> 케타민 (80mg/kg)과 xylazine (8mg/kg) (2시 1분에서 볼륨 비율)의 내부 복막 주사로 쥐 마취시키다. </li><li> intravitreous 주입하기 전에 국소 anesthetics으로 쥐 눈 0.5 % alcaine 한방울을 적용합니다. </li><li> 쥐 위치와 안구의 우수한 비강 지역을 쉽게받을 수 있습니다. </li><li> 파 플라나의 수준에 찔린 우수한 비강 공막엔를 30 게이지 바늘을 사용합니다. 안구 근육과 혈관을 만지는 것을 피하십시오. </li><li> 뒷부분 챔버에서 바늘 구멍을 통해 유리의 작은 금액을 없애 눈에 볼 가벼운 압력을주세요. </li><li> 유리체 몸으로 공막엔을 통해 45 ° 각도로 바늘 구멍을 통해 유리 피펫의 팁을 넣습니다. 후부 챔버로 솔루션 2 ML을 주사하고 몇 초 약 장소에 보관 후, 부드럽게 바늘을 제거합니다. 렌즈 intraocular 선박을 만지는 것을 피하십시오. </li><li> intravitreous 주입 후 감염을 방지하기 위해 쥐의 안구에 안과 Tobrex의 연고를 적용합니다. </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>

동물에 대한 비전과 안과 제표 연구에 대한 홍콩의 대학과 협회의 교육 및 연구에 살아있는 동물의 사용 (CULATR)의위원회에 의해 승인된 연구에 동물의 사용을 위해 프로토콜에 따라 처리되었습니다 안과 및 비전 연구에 동물 사용합니다.

좋은 intravitreous 사출 직접 intraocular 치료뿐만 아니라, 동물 모델을 수립하는 데 유용합니다. 첫째, 주사 사이트는 망막 구조를 중단해서는 안됩니다. 둘째, 출혈은 감염의 위험을 줄이기 위해 피해야한다. 셋째, 렌즈 외상성 백내장을 피하기로 온전히해야합니다. 가장 중요한 것은 정상적인 안구 구조의 중단을 줄일 수 있습니다.

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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

Intravitreous 주사는 시각 과학 연구에 널리 사용되는 기법입니다. 이것은 안구 질환이나 현지 치료의 직접적인 응용 프로그램으로 동물 모델을 확립하는 데 사용할 수 있습니다. 이 동영상은 intravitreous 사출 절차를 완료하기 위해 간단하고 저렴한 도구를 사용하는 방법을 소개합니다. 대신 hemilton의 주사기의 1 ML 주사기의 사용이 사용됩니다. 적절한 사출 완벽한 팁을 유리 pipettes를 사용하는 바늘, 얼마나 쉽게 단단하게 함께 유리 피펫과 주사기 바늘을 연결을하는 방법에 대한 실용 팁이 제공됩니다. 한 주사 사이트는 망막 구조를 방해해서는 안) 2) 출혈은 감염의 위험을 줄이기 위해 피해야한다, 3) 렌즈는 외상 백내장을 피하기 위해 온전히되어야합니다 좋은 intravitreous 주사를 실시하기 위해 관찰하는 세 가지 측면이 있습니다. 간단히, 가장 중요한 포인트는 일반적인 안구 구조의 중단을 줄일 수 있습니다. 망막의 중단을 방지하려면, 쥐 눈 우수한 비강 지역은 선정되었습니다. 또한, 바늘의 바늘 포인트는 쥐 눈 limbal 지역에서 약 1.5 mm 있었던 파 평면에서했다. 유리체의 소량을 부드럽게 주입하기 전에 intraocular 압력을 줄이기 위해 바늘 구멍을 통해 밖으로 밀고있다. 45 ° 분사 각도로, 따라서 렌즈형 요인의 관련 합병증 및 영향을 피하기 위해, 쥐의 안구에서 외상성 백내장을 일으킬 가능성이 적습니다. 이 작업에서는 결막과 안구 근육에 대한 절단, 아니 출혈이 없었습니다. 신속하고 가벼운 부상으로 성공적인 intravitreous 분사는 분 안에 수행할 수 있습니다. 사출이 특정 프로토콜에 설명된 설정하면 intravitreous 주사에만 적용됩니다. 그러나, 여기에 제시된 방법과 자료는 또한 뇌, 척수 또는 작은 포유류의 다른 장기에 약물 전달의 다른 주입 절차에 사용할 수 있습니다.

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

Intravitreous Injection for Establishing Ocular Diseases Model

Intravitreous 주사는 안구 질환 또는 현지 치료의 직접적인 응용 프로그램으로 시각 과학 연구에 널리 사용되는 기법입니다. 이 동영상은 유리 피펫과 1ml 주사기를 사용하여 intravitreous 사출을위한 프로토콜을 보여주었다. 과다 출혈 및 렌즈 손상을 피하에 대한 유용한 팁은 주어집니다.

안구 질환 모델 구축을위한 Intravitreous 사출

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 조회수.  The University of Hong Kong - HKU. Intravitreous 주사는 안구 질환 또는 현지 치료의 직접적인 응용 프로그램으로 시각 과학 연구에 널리 사용되는 기법입니다. 이 동영상은 유리 피펫과 1ml 주사기를 사용하여 intravitreous 사출을위한 프로토콜을 보여주었다. 과다 출혈 및 렌즈 손상을 피하에 대한 유용한 팁은 주어집니다.

비디오: 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.

SD 쥐에서 레이저 유도 만성 안구 고혈압 모델

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

연구

생물학

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.

인터뷰 : 단백질 폴딩과 Neurodegenerative 질병의 연구

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.

Zebrafish의 두뇌 뇌실 주입

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.

눈 Microvessels의 질량 분석 기반 Proteomics 분석을 위한 시료 준비

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.

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.

카노르하비티스 예르간에서 노화와 관련된 신경 퇴행성 질환 모델링

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.

통합 된 라이브 셀 모니터링을 통해 인간 유도 만능 줄기 세포 유래 피질 및 도파민 뉴런의 자동화 된 생산

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.

안구 미세 혈관 신생의 전 비보 코로이드 발아 분석

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.

1차 마우스 망막 색소 상피 세포의 효율적인 해부 및 배양

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.

일차 및 불멸화 된 인간 각막 상피 세포에 대한 UV 방사선 및 화학 물질의 독성 결정

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