Name: high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury
Uploaded: 2018-06-16T13:00:00.000+00:00
Duration: 14 min 20 s
Description: Watch this Scientific Journal Video about High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury at JoVE.com

이 연구로는 국립 연구소의 건강 중화 프로그램 부처간 계약 # AOD13015-001 지원 되었다. 우리는 그들의 노력과 전문 비디오 제작에 대 한 스테파니 Froberg 피터 허스트를 감사 하 고 싶습니다.

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저자는 공개 없다.
면책 조항:이 문서에 표현 들 저자와 군대의 부, 국방부, 또는 미국 정부의 공식 정책 반영 하지 않습니다. 이 연구는 NIH/NIAID 사이의 USAMRICD, 부처간의 합의 의해 지원 되었고 일부 대학원 연구 참여 프로그램에는 미국 육군 의료 연구 연구소의 화학 방어에 약속에 의해 오크에 의해 관리 릿지 연구소 과학 미국 에너지 부와 USAMRMC 사이의 부처간 합의 통해 교육에 대 한.

본 모델의 HF와 CP 부상 연구에 대 한 심사는 높은 처리량 각 막 상피 세포의 개발에 우리의 방법 및 결과 설명 우리. 우리는 또한 HF 부상에 대 한 기본 siRNA 화면에서 결과 제시. TIC 상해 연구를 위한 HTS 모델의 개발에 많은 도전을 했다. 우리가 셀 문화 모델에 HF, HFA 또는 CP의 연구와 관련 된 문학에서 찾을 수 있는 방법은 작은 도움의 이었다. 불 소 이온에 대부분 생체 외에서 연구 구강 세포?...

Toxicant 유도 눈 부상 화학 빠르게 상당한 조직 손상을 입힐 잠재력을가지고 있기 때문에 진정한 눈 비상 이다. 불행 하 게도, 아니 특정 치료제가이 부상 치료 존재에 toxicant 유발 각 막 부상에 대 한 치료 일반적으로 지원만 있습니다. 현재 치료 전략 일반적인 주로 같은 윤활제, 항생제, 국 소 치료 요법을 포함 하 고 cycloplegics 뒤에 소염제 (예를 들어, 스테로이드) 한 번 각 막1 epithelialized 다시 있다 ,2. 최고의 현재 치료 치료 옵션을 사용할에 불구 하 고 장기적인 예 후는 일반적으로 진보적인 각 막 흐려 및 neovascularization2,3가난.
동물 모델 화학 독성을 조사 하 고 상해의 메커니즘을 이해를 전통적으로 사용 되어 왔습니다. 그러나, 동물 연구는 시간이 소모 되 고 비싸다. 노력을 줄이기 위해 동물 실험도 있다. 예를 들어 유럽 연합에 도달 법안 (EC 1907/2006)를 동물 실험을 줄이기 위한 규정 있다. 규정 회사 동물 테스트 및 동물에 제안 된 테스트를 수행 하기 전에 유럽 화학 제품 기관에서 승인을 얻는 피하기 위하여 데이터 공유 요구 사항이 포함 됩니다. 규정에 따라 동물 실험 최후의 수단 이어야 한다. 또한 단계적으로 동물에서 화장품의 테스트 유럽 화장품 규정 (EC 1223/2009)이 있다. 동물 연구를 실시 하는 경우 그들이 3Rs의 원칙에 의해 인도 된다 (구체화, 감소, 및 교체), 더 자비 롭 동물 연구를 수행, 사용, 동물의 수를 감소 및 비 동물 대안을 사용 하 여 프레임 워크를 제공 하는 가능한 경우. 이러한 이유로 독물학 분야는 독성의 분자 메커니즘에 대 한 통찰력을 제공할 수 있습니다 하 고 높은 처리량4에서 행 해질 수 있다 생체 외에서 분석을 채택 하고자 결정 했다. 이것은 기능 독극물 접근 어디 유독 전적으로 그들의 화학에 의해 보다는 오히려 그들의 기능에 의해 정의 됩니다. 단계는 또한, 기능 toxicogenomics 추구 특정 유전자 유독5의 효과에서 재생 역할을 이해 하. SiRNA 기술을 응용, 유독에 분자와 세포질 응답에 유전자 기능을 조사 하기 위해 화면 높은 처리량에 수행할 수 있습니다. siRNA는 19-25의 뉴클레오티드 긴 포스트 transcriptional 유전자 입을 통로 모든 포유류 세포6에 활용 하는 두 배 좌초 된 RNA 분자. 이 종합적으로 만들어지고 특정 유전자를 대상으로 설계 된. 셀에 도입 하는 경우는 siRNA 처리 되 고 한 가닥, 가이드 물가 RNA 유도 입을 복잡 한 (RISC)에 로드 됩니다. siRNA는 mRNA 분자에 상호 보완적인 영역에 RISC 지시 하 고 있는 RISC 저하는 mRNA. 특정 유전자의 표정 감소 결과. 특정 유전자의 표현의 결과 감소는 toxicant에 세포질 응답에 그 유전자의 기능을 확인 toxicant 노출된 셀에 다음 공부 수 있습니다. 이러한 접근은 더욱 CYP1A17,8의 a h R-종속 유도 리 민감성의 메커니즘 이해 하 사용 되었습니다.
화학 테러 위험 평가 (CTRA) 목록 및 독성 산업용 화학 물질 (TIC) 목록 선택 화학 물질의 독성과 테러, 전쟁, 또는 산업 재해 이벤트9중 출시 될 가능성에 따라 항목별로 나 누이고 있다. 우리는 siRNA 높은 처리량 검열 (HTS) toxicogenomic 접근 테러 사건에서 사용의 높은 위험에 확인 된 CTRA 목록 이용과의 연구에 적용 하 고 있다. 화학 생물;에 있는 불리 한 효과 이해 하고자 하는 전통적인 독물학 그러나, 우리는 더 욕망의 치료제 및 치료 접근을 가능 하 게, 개발을 알리는 목적으로 상해의 메커니즘을 이해 하는 분자 치료 개발에 대 한 대상으로 지정할 수 있는 발견. 몇 가지 방법으로이 노력 유사한 높은 처리량 siRNA 심사 및 약 발견 과정10에서 셀 기반 분석의 사용을 고려할 수 있습니다. 주요 차이점은 우리의 접근 방식에 다소 가능성이 크다 toxicant 노출의 치료에 대 한 높은 치료 가치와 단일 대상 될 것 이다 반면 그 약물 발견 일반적으로 치료 발견에 대 한 단 수 목표를 추구 될 것 이다. 우리 예상 어떤 효과적인 치료 패러다임 toxicant 노출에 대 한 높은 치료 가치를 달성 하는 다각적인 접근 방식이 필요로 하 고 toxicogenomic 데이터 극히 효과적인 치료 패러다임을 알릴 수 있습니다.
벤치탑 자동화 실험실 제약 또는 생명 공학 산업 밖에 서 높은 처리량 방법론을 제공합니다. 우리 연구소에서 생체 외에서 연구 역사적으로 낮은 처리량11,,1213전통적인 분석 되었습니다. 지난 몇 년 동안, 우리의 실험실 벤치탑 높은 처리량 siRNA 심사를 수행 하기 위해 로봇의 사용에 전환 했다. 여기, 우리가 현재 눈 셀 모델의 세련미와 체 외에서 의 개발 수소 불 화물 (HF)와 chloropicrin (CP) 높은 처리량 siRNA 심사 적합에 대 한 노출 방법. 우리의 목표는 이러한 유독 응답에 세포질 상해를 조절 하는 분자를 식별 하는. 우리는 선택 하는 siRNA 도서관의 목표는 G 단백질 결합 된 수용 체, 단백질 kinases, 프로 테아 제, 가수분해, 이온 채널, 그리고 다른 잠재적으로 druggable 대상 포함 됩니다. HF와 CP 선정 됐다 연구에 대 한 상호 CTRA 목록 에이전트 산업 사고의 ToxNet 보고서를 통해 증기 노출9,14눈 상해의 가장 중대 한 모험을 선물 하는 그를 찾을 수 있습니다. WWI15에 최 루 가스 CP (Cl3CNO2화학식, CAS 번호 76-06-2) 원래 사용 했다. 현재16nematicide, 살 균 제, 살충제는 농업 fumigant 및 기능으로 사용 됩니다. 수소 불 화물 (HF)는 정유 및 화학 fluorination 유기 화합물17의 알을 포함 하 여 프로세스에서 사용 됩니다. HF (화학식 HF, CAS 번호 62778-11-4)는 가스 이지만 수성 형태로 소산 (HFA, CAS 번호 7664-39-3). 따라서, 우리가 우리의 셀 HFA를 사용 하 여 선출 노출 모델. SV40 큰 T 항 원 SV40 HCEC 연구에 대 한 선정 되었다 인간 각 막 상피 세포 선 불후. 세포질 상해에 관련 된 목표 세포 죽음과 선 동적인 응답에 반영 해야 하기 때문에 세포 생존 능력 및 염증 성 마커 IL-8 끝점으로 선정 됐다. 특히, 대상 toxicant 노출에서 보호 역할을 한다면, 세포 죽음 또는 염증 성 cytokine 생산 증가 한다 때 대상 식 siRNA에 의해 저해. 반대로 부정적인 역할을 하는 대상에 대 한 사실 것입니다. 또한, 만성 염증 후 노출, 및 세포 죽음 통로 있는 내정간섭에 임상 결과2,18이 향상 시킬 수 있습니다 각 막 병 리에 역할을 나타납니다.

<table><tbody><tr><td>Bravo liquid handing platform</td><td>Agilent or equivalent</td><td>G5409A</td><td></td></tr><tr><td>Bravo plate shaker</td><td>Agilent or equivalent</td><td>Option 159</td><td></td></tr><tr><td>Bravo 96LT disposable tip head</td><td>Agilent or equivalent</td><td>Option 178</td><td>96-channel large tip pipetting head unit</td></tr><tr><td>Bravo 96ST disposable tip head</td><td>Agilent or equivalent</td><td>Option 177</td><td>96-channel small tip pipetting head unit</td></tr><tr><td>Bravo 384ST disposable tip head</td><td>Agilent or equivalent</td><td>Option 179</td><td>384-channel small tip pipetting head unit</td></tr><tr><td>Bravo 96 250 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19477-022</td><td></td></tr><tr><td>Bravo 384 30 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19133-212</td><td></td></tr><tr><td>Bravo 384 70 &amp;mu;L sterile barrier tips</td><td>Agilent or equivalent</td><td>19133-212</td><td></td></tr><tr><td>EnSpire multimode plate reader</td><td>Perkin Elmer or equivalent</td><td>2300-0000</td><td>AlphaLISA assay detector with high power laser excitation</td></tr><tr><td>IL-8 (human) AlphaLISA Detection Kit&amp;nbsp;</td><td>Perkin Elmer or equivalent</td><td>AL224F</td><td>no-wash bead-based assay</td></tr><tr><td>ProxiPlate-384 Plus white 384-shallow well microplates</td><td>Perkin Elmer or equivalent</td><td>6008359</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Invitrogen or equivalent</td><td>13778500</td><td>Transfection reagent</td></tr><tr><td>Opti-MEM 1 Reduced Serum Medium</td><td>Invitrogen or equivalent</td><td>31985070</td><td></td></tr><tr><td>TrypLE Express</td><td>Gibco or equivalent</td><td>12605010</td><td>Cell detachment solution</td></tr><tr><td>IncuCyte Zoom</td><td>Essen Instruments or equivalent</td><td>ESSEN BIOSCI 4473</td><td>Incubator-housed automated microscope</td></tr><tr><td>Chloropicrin</td><td>Trinity Manufacturing or equivalent</td><td>N/A</td><td>Acute toxicity and irritant</td></tr><tr><td>DMEM-F12 cell culture medium</td><td>Invitrogen or equivalent</td><td>11330-057</td><td>Contains HEPES</td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen or equivalent</td><td>1891471</td><td></td></tr><tr><td>Human epidermal growth factor (cell culture grade)</td><td>Invitrogen or equivalent</td><td>E9644-.2MG</td><td></td></tr><tr><td>Recombinant human insulin (cell culture grade)</td><td>Invitrogen or equivalent</td><td>12585-014</td><td></td></tr><tr><td>Penicillin-Streptomycin solution (cell culture grade)</td><td>Invitrogen or equivalent</td><td>15140122</td><td></td></tr><tr><td>Hydrocortisone (cell culture grade)</td><td>Sigma or equivalent</td><td>H0888-10G</td><td></td></tr><tr><td>Glucose&amp;nbsp; (cell culture grade)</td><td>Sigma or equivalent</td><td>G7021</td><td></td></tr><tr><td>PBS&amp;nbsp; (cell culture grade)</td><td>Sigma or equivalent</td><td>P5493</td><td></td></tr><tr><td>siRNA</td><td>Dharmacon or equivalent</td><td>various</td><td></td></tr><tr><td>Thiazolyl blue tetrazolium bromide</td><td>Sigma or equivalent</td><td>M5655</td><td>MTT assay substrate</td></tr><tr><td>siRNA buffer</td><td>Thermo or equivalent</td><td>B002000</td><td></td></tr><tr><td>96-well cell culture plates</td><td>Corning or equivalent</td><td>CLS3595</td><td></td></tr><tr><td>T150 cell culture flasks</td><td>Corning or equivalent</td><td>CLS430825</td><td></td></tr><tr><td>BSL-2 cell culture hood</td><td>Nuaire or equivalent</td><td>NU-540</td><td></td></tr><tr><td>300 mL robotic reservoirs</td><td>Thermo or equivalent</td><td>12-565-572&amp;nbsp;</td><td></td></tr><tr><td>96 baffled automation reservoirs</td><td>Thermo or equivalent</td><td>1064-15-8</td><td></td></tr><tr><td>500 mL sterile disposable storage bottles</td><td>Corning or equivalent</td><td>CLS430282</td><td></td></tr><tr><td>Microplate heat sealer</td><td>Thermo or equivalent</td><td>AB-1443A</td><td></td></tr><tr><td>Microplate heat sealing foil</td><td>Thermo or equivalent</td><td>AB-0475</td><td></td></tr><tr><td>Cardamonin</td><td>Tocris or equivalent</td><td>2509</td><td>Anti-inflammatory, used as positive control</td></tr><tr><td>SKF 86002&amp;nbsp;</td><td>Tocris or equivalent</td><td>2008</td><td>Anti-inflammatory, used as positive control</td></tr><tr><td>DMSO</td><td>Sigma or equivalent</td><td>D8418</td><td></td></tr><tr><td>48% hydrofluoric acid</td><td>Sigma or equivalent</td><td>339261</td><td>Corrosive and acute toxicity</td></tr><tr><td>1000 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014382</td><td></td></tr><tr><td>200 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014391</td><td></td></tr><tr><td>20 &amp;mu;L Single channel pipettors</td><td>Rainin or equivalent</td><td>17014392</td><td></td></tr><tr><td>1000 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17014497</td><td></td></tr><tr><td>200 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17013810</td><td></td></tr><tr><td>20 &amp;mu;L 12-channel pipettors</td><td>Rainin or equivalent</td><td>17013808</td><td></td></tr><tr><td>Pipettor tips 1000 &amp;mu;L</td><td>Rainin or equivalent</td><td>17002920</td><td></td></tr><tr><td>Pipettor tips 200 &amp;mu;L</td><td>Rainin or equivalent</td><td>17014294</td><td></td></tr><tr><td>Pipettor tips 20 &amp;mu;L</td><td>Rainin or equivalent</td><td>17002928</td><td></td></tr><tr><td>Chemical fume hood</td><td>Jamestown Metal Products</td><td>MHCO_229</td><td></td></tr><tr><td>384-well sample storage plates</td><td>Thermo or equivalent</td><td>262261</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma or equivalent</td><td>S6191</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Thermo or equivalent</td><td>14-959-49A</td><td></td></tr><tr><td>Serological pipettes 50 mL</td><td>Corning or equivalent</td><td>07-200-576</td><td></td></tr><tr><td>Serological pipettes 25 mL</td><td>Corning or equivalent</td><td>07-200-575</td><td></td></tr><tr><td>Serological pipettes 10 mL</td><td>Corning or equivalent</td><td>07-200-574</td><td></td></tr><tr><td>Serological pipettes 5 mL</td><td>Corning or equivalent</td><td>07-200-573</td><td></td></tr><tr><td>SV40-HCEC immortalized human corneal epithelial cells</td><td>N/A</td><td>N/A</td><td>These cells are not commercially available, but can be obtained from the investigators cited in the article</td></tr><tr><td>Sceptor Handheld Automated Cell Counter</td><td>Millipore or equivalent</td><td>PHCC20060</td><td></td></tr><tr><td>GeneTitan Multi-Channel (MC) Instrument</td><td>Affymetrix or equivalent</td><td>00-0372</td><td></td></tr><tr><td>Affymetrix 24- and 96-array plates</td><td>Affymetrix or equivalent</td><td>901257; 901434</td><td></td></tr><tr><td>Draegger tube HF</td><td>Draeger or equivalent</td><td>8103251</td><td></td></tr><tr><td>Draegger tube CP</td><td>Draeger or equivalent</td><td>8103421</td><td></td></tr><tr><td>Draegger pump</td><td>Draeger or equivalent</td><td>6400000</td><td></td></tr><tr><td>Clear Plate seals</td><td>Resesarch Products International or Equivalent</td><td>202502</td><td></td></tr><tr><td>Reagent reservoirs</td><td>VistaLab Technologies or equivalent</td><td>3054-1000</td><td></td></tr><tr><td>Xlfit</td><td>IDBS or equivalent</td><td>N/A</td><td>Excel add-in used for automated curve fitting</td></tr></tbody></table>

high throughput sirna screening for chloropicrin and hydrogen fluoride-induced cornea epithelial cell injury

Toxicant 유도 눈 부상 화학 빠르게 상당한 조직 손상을 입힐 잠재력을가지고 있기 때문에 진정한 눈 비상 이다. 아무 특정 치료제가이 부상 치료 존재 toxicant 유발 각 막 부상에 대 한 치료는 일반적으로 지원. 치료 및 노출에 대 한 관심에 치료제를 개발 하는 노력, 그것은이 상해의 분자 및 세포 메커니즘을 이해 하는 것이 중요 수. 있습니다 우리는 높은 처리량 작은 금지 RNA (siRNA) 심사의 활용 화학 각 막 상피 상해의 분자 메커니즘을 더 빠르게 명료를 도울 수 있는 중요 한 도구가 될 수 있습니다 제안 합니다. siRNA는 siRNA에 상 동을가지고 있는 19-25의 뉴클레오티드 긴 mRNA를 저하 하는 통로 입을 post-transcriptional 유전자를 활용 더블 좌초 RNA 분자는. 특정 유전자의 표현의 결과 감소는 toxicant에 세포질 응답에 그 유전자의 기능을 확인 toxicant 노출된 셀에 다음 공부 수 있습니다. 개발 및 체 외에 노출 모델 및 수소 불 화물 (HF) 및 chloropicrin (CP) 유도 눈 부상 (HTS)을 상영 하는 높은 처리량 방법의 유효성 검사는이 문서에 표시 됩니다. 하지만 우리는 이러한 두 가지 유독 선택, 우리의 방법 toxicant 노출 프로토콜에 사소한 수정 다른 유독의 연구에 적용 됩니다. SV40 큰 T 항 원 SV40 HCEC 연구에 대 한 선정 되었다 인간 각 막 상피 세포 선 불후. 세포 생존 능력 및 IL-8 생산 심사 프로토콜에서 끝점으로 선정 됐다. Toxicant 노출의 개발와 관련 된 몇 가지 과제 고 셀 문화 방법 HTS 연구에 대 한 적합 한 표시 됩니다. 이 유독 HTS 모델 설립 화학 눈 부상에 대 한 잠재적인 치료에 대 한 화면을 상해의 메커니즘을 이해 하기 더 연구에 대 한 수 있습니다.

노출 방법 개발
우리는 세련 하 고 HTS 연구에 사용 하기 위해 인간 각 막 상피 세포 선 SV40-HCEC의 적합성을 평가. SV40 HCEC SV 40 큰 T 항 원을 사용 하 여 불멸 하 게 했다 고 Dhanajay Pal23에서 선물 했다. 간결 하 게, 여기 제시 노출 방법론 개발에 탐구 하는 너무 많은 변수가 있었고 그래서, 우리가 생각 ...

Watch this Scientific Journal Video about High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury at JoVE.com

High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

높은 처리량 작은 금지 RNA 검사 화학 각 막 상피 상해의 분자 메커니즘을 더 빠르게 명료를 도울 수 있는 중요 한 도구입니다. 여기, 우리는 개발 및 노출 모델 및 각 막 상피 부상 수소 불 화물 그리고 chloropicrin 유도의 높은 처리량 검열 방법의 유효성 검사 제시.

Chloropicrin 및 수소 불 화물 유발 각 막 상피 세포 상해에 대 한 높은 처리량 SiRNA 심사

Toxicant-induced ocular injury is a true ocular emergency because chemicals have the potential to rapidly inflict significant tissue damage. Treatments ...

high-throughput-sirna-screening-for-chloropicrin-hydrogen-fluoride

Research

JoVE Journal

Biology

5.8K 조회수.  US Army Medical Research Institute of Chemical Defense. 높은 처리량 작은 금지 RNA 검사 화학 각 막 상피 상해의 분자 메커니즘을 더 빠르게 명료를 도울 수 있는 중요 한 도구입니다. 여기, 우리는 개발 및 노출 모델 및 각 막 상피 부상 수소 불 화물 그리고 chloropicrin 유도의 높은 처리량 검열 방법의 유효성 검사 제시.

비디오: High Throughput SiRNA Screening for Chloropicrin and Hydrogen Fluoride-Induced Cornea Epithelial Cell Injury

RNA Interference-based Investigation of the Function of Heat Shock Protein 27 during Corneal Epithelial Wound Healing

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex created to specifically target a mRNA transcript to induce its degradation and it has been used to identify novel pathways in various cellular processes. Few reports exist regarding the role of phosphorylated heat shock protein 27 (HSP27) in corneal epithelial wound healing. Herein, cultured human corneal epithelial cells were divided into a scrambled control-siRNA transfected group and a HSP27-specific siRNA-transfected group. Scratch-induced directional wounding assays, and western blotting, and flow cytometry were then performed. We conclude that HSP27 has roles in corneal epithelial wound healing that may involve epithelial cell apoptosis and migration. Here, step-by-step descriptions of sample preparation and the study protocol are provided.

Herein, we present a protocol to use heat shock protein 27 (HSP27)-specific small interfering RNA to assess the function of HSP27 during corneal epithelial wound healing. RNA interference is the best method for effectively knocking-down gene expression to investigate protein function in various cell types.

각막 상피 ​​창상 치유 기간 동안 열 충격 단백질 27의 기능의 RNA 간섭 기반 조사

Small interfering RNA (siRNA) is among the most widely used RNA interference methods for the short-term silencing of protein-coding genes. siRNA is a ...

연구

유전학

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

생체공학

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

자외선-C (UV-C) 손상에 응하여 1 차 마우스 안구 표면 세포/줄기 세포에 있는 산화 손상의 평가

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

발생학

High-throughput Measurement of Plasma Membrane Resealing Efficiency in Mammalian Cells

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In response to these damages, complex molecular machineries rapidly reseal the plasma membrane to restore its barrier function and maintain cell survival. Despite 60 years of research in this field, we still lack a thorough understanding of the cell resealing machinery. With the goal of identifying cellular components that control plasma membrane resealing or drugs that can improve resealing, we have developed a fluorescence-based high-throughput assay that measures the plasma membrane resealing efficiency in mammalian cells cultured in microplates. As a model system for plasma membrane damage, cells are exposed to the bacterial pore-forming toxin listeriolysin O (LLO), which forms large 30-50 nm diameter proteinaceous pores in cholesterol-containing membranes. The use of a temperature-controlled multi-mode microplate reader allows for rapid and sensitive spectrofluorometric measurements in combination with brightfield and fluorescence microscopy imaging of living cells. Kinetic analysis of the fluorescence intensity emitted by a membrane impermeant nucleic acid-binding fluorochrome reflects the extent of membrane wounding and resealing at the cell population level, allowing for the calculation of the cell resealing efficiency. Fluorescence microscopy imaging allows for the enumeration of cells, which constitutively express a fluorescent chimera of the nuclear protein histone 2B, in each well of the microplate to account for potential variations in their number and allows for eventual identification of distinct cell populations. This high-throughput assay is a powerful tool expected to expand our understanding of membrane repair mechanisms via screening for host genes or exogenously added compounds that control plasma membrane resealing.

Here we describe a high-throughput fluorescence-based assay that measures the plasma membrane resealing efficiency through fluorometric and imaging analyses in living cells. This assay can be used for screening drugs or target genes that regulate plasma membrane resealing in mammalian cells.

포유류 세포에서 효율 Resealing 원형질 막의 높은 처리량 측정

In their physiological environment, mammalian cells are often subjected to mechanical and biochemical stresses that result in plasma membrane damage. In ...

면역학 및 감염병학

간암 세포에서 Dihydroethidium을 사용한 새로운 ROS 정량 분석

High Throughput Screening Assessment of Reactive Oxygen Species (ROS) Generation using Dihydroethidium (DHE) Fluorescence Dye

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS production plays a central role in the spatial and temporal modulation of normal cellular functions such as proliferation, signaling, apoptosis, and senescence. In contrast, chronic ROS overproduction is responsible for a wide spectrum of diseases, such as cancer, cardiovascular disease, and diabetes, among others. Quantifying ROS levels in an accurate and reproducible manner is thus essential to understanding normal cellular functionality. Fluorescence imaging-based methods to characterize intra-cellular ROS species is a common approach. Many of the imaging ROS protocols in the literature use 2&#39;-7&#39;-dichlorodihydrofluorescein diacetate (DCFH-DA) dye. However, this dye suffers from significant limitations in its usage and interpretability. The current protocol demonstrates the use of a dihydroethidium (DHE) fluorescent probe as an alternative method to quantify total ROS production in a high-throughput setting. The high throughput imaging platform, CX7 Cellomics, was used to measure and quantify the ROS production. This study was conducted in three hepatocellular cancer cell lines - HepG2, JHH4, and HUH-7. This protocol provides an in-depth description of the various procedures involved in the assessment of ROS within the cells, including - preparation of DHE solution, incubation of cells with DHE solution, and measurement of DHE intensity necessary to characterize the ROS production. This protocol demonstrates that DHE fluorescent dye is a robust and reproducible choice to characterize intracellular ROS production in a high-throughput manner. High throughput approaches to measure ROS production are likely to be helpful in a variety of studies, such as toxicology, drug screening, and cancer biology.

저자 스포트라이트: 간세포주에서 세포 내 ROS 수준의 고처리량 측정

This protocol describes a novel method to quantify intracellular reactive oxygen species (ROS) using dihydroethidium (DHE) as a fluorescence dye probe using a high-throughput screening approach. The protocol describes the methods for quantitative assessment of intracellular reactive oxygen species (ROS) in the three different hepatocellular carcinoma cell lines.

Dihydroethidium(DHE) 형광 염료를 사용한 활성 산소종(ROS) 생성의 고처리량 스크리닝 평가

Reactive oxygen species (ROS) play a key role in the regulation of cellular metabolism in physiological and pathological processes. Physiological ROS ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

생물학

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it is challenging to assess the contributions of individual constituents to its overall toxicity. Toxicological profiles of individual constituents as well as mixtures can be however established in vitro, by applying high through-put screening tools, which enable the profiling of Harmful and Potentially Harmful Constituents (HPHCs) of tobacco smoke, as defined by the U.S. Food and Drug Administration (FDA).2
For an initial assessment, an impedance-based instrument was used for a real-time, label-free assessment of the compound&#39;s toxicity. The instrument readout relies on cell adhesion, viability and morphology that all together provide an overview of the cell status. A dimensionless parameter, named cell index, is used for quantification. A set of different staining protocols was developed for a fluorescence imaging-based investigation and a HCS platform was used to gain more in-depth information on the kind of cytotoxicity elicited by each HPHC.
Of the 15 constituents tested, only five were selected for HCS-based analysis as they registered a computable LD50 (&#60; 20 mM). These included 1-aminonaphtalene, Arsenic (V), Chromium (VI), Crotonaldehyde and Phenol. Based on their effect in the HCS, 1-aminonaphtalene and Phenol could be identified to induce mitochondrial dysfunction, and, together with Chromium (VI) as genotoxic based on the increased histone H2AX phosphorylation. Crotonaldehyde was identified as an oxidative stress inducer and Arsenic as a stress kinase pathway activator.
This study demonstrates that a combination of impedance-based and HCS technologies provides a robust tool for in vitro assessment of CS constituents.

High Content Screening Analysis to Evaluate the Toxicological Effects of Harmful and Potentially Harmful Constituents HPHC

The objective of the study was to assess the biological impact of 15 cigarette smoke constituents using a combination of an impedance-based real time cell analyzer and a high-content screening (HCS)-based platform for toxicological assessment in vitro. This study provides information on effective doses, toxicity and modes of action of the tested compounds.

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

Cigarette smoke (CS) is a major risk factor for cardiovascular and lung diseases. Because CS is a complex aerosol containing more than 7,000 chemicals1 it ...

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) ...

Chloropicrin 및 수소 불 화물 유발 각 막 상피 세포 상해에 대 한 높은 처리량 SiRNA 심사

요약

더 많은 비디오 탐색

이 비디오의 챕터

각막 상피 창상 치유 기간 동안 열 충격 단백질 27의 기능의 RNA 간섭 기반 조사

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

자외선-C (UV-C) 손상에 응하여 1 차 마우스 안구 표면 세포/줄기 세포에 있는 산화 손상의 평가

포유류 세포에서 효율 Resealing 원형질 막의 높은 처리량 측정

Dihydroethidium(DHE) 형광 염료를 사용한 활성 산소종(ROS) 생성의 고처리량 스크리닝 평가

Dihydroethidium(DHE) 형광 염료를 사용한 활성 산소종(ROS) 생성의 고처리량 스크리닝 평가

Dihydroethidium(DHE) 형광 염료를 사용한 활성 산소종(ROS) 생성의 고처리량 스크리닝 평가

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

유해 및 잠재적으로 유해한 구성 성분의 독성 효과를 평가하기 위해 높은 콘텐츠 심사 분석 (HPHC)

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

Chloropicrin 및 수소 불 화물 유발 각 막 상피 세포 상해에 대 한 높은 처리량 SiRNA 심사

요약

더 많은 비디오 탐색

이 비디오의 챕터

각막 상피 ​​창상 치유 기간 동안 열 충격 단백질 27의 기능의 RNA 간섭 기반 조사

복원 된 인간의 각막 모양의 상피 (RHCE) 조직 모델을 사용하여 눈에 자극성 화학 물질의 위험 식별을위한 눈 자극 시험 (EIT)

자외선-C (UV-C) 손상에 응하여 1 차 마우스 안구 표면 세포/줄기 세포에 있는 산화 손상의 평가

포유류 세포에서 효율 Resealing 원형질 막의 높은 처리량 측정

Dihydroethidium(DHE) 형광 염료를 사용한 활성 산소종(ROS) 생성의 고처리량 스크리닝 평가

Dihydroethidium(DHE) 형광 염료를 사용한 활성 산소종(ROS) 생성의 고처리량 스크리닝 평가

Dihydroethidium(DHE) 형광 염료를 사용한 활성 산소종(ROS) 생성의 고처리량 스크리닝 평가

후 전사적으로 규제 유전자의 화학 변조기에 대한 높은 처리량 검사

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