우리는 DRS 감사합니다. 존 핸콕, 레이 그릴, 앤드류 모리스과 지원 올가 Chumakova. FlexStation 3 시스템은 UTHSC Cytodynamic 이미징 시설의 일부입니다. 우리는 또한 DRS 감사합니다. 스크립스 연구소와 HEK293 - hTRPA1 안정 세포주를 제공 노바티스 연구 재단 (GNF)의 게놈 연구소에서 Ardem Patapoutian 마이클 Bandell. 연구는 NIH (MXZ에 GM081658)와 텍사스 메디컬 센터 소화기 질환 센터 (HH)을하고 미션 연결 / TIRR 재단 (HH로)에서 부여로 일부에서 지원됩니다.

1. 96 - 웰 플레이트에 셀 준비 transiently cDNA 인간 TRPA1와 trasfected HEK293 세포에 대한 <ol><li> HEK293 세포는 10 %의 열 inactivated 태아 소 혈청 (Invitrogen, 16000-044)와 보충 4.5 MG / ML 포도당 (써모 과학, SH3002201)이있는 Dulbecco의 최소 필수 매체 1~2일 (DMEM), 50 단위 재배 / ML 페니실린 50 μg / ML 스트렙토 마이신 (Invitrogen, 15140-163). transfection의 날, 세포 밀도는 70-90%에 도달해야합니다. </li><li> 각 물론 50 μl 폴리 - L - ornithine (시그마 P3655, 멸균 증류수에 20 μg / ML)를 pipetting 37에서 잠복기 ° C를 최소한 15 분에 의해 외투 96 - 웰 플레이트. Dulbecco의 인산 각각 잘 한 번 씻어도 MG이없이 살린 (DPBS)를 버퍼 + 및 CA 2 + (써모 과학, SH3002803). 번호판은 몇 시간 동안 세포 배양 후드에 남아있을 수 있습니다. </li><li> 96 - 웰 플레이트 모든 잘 들어, 1.5 ML Eppendorf 튜브 25 μl OPTI - 멤 (Invitrogen, 31985-070)의 cDNA 0.2 μg에 희석. 동일한 cDNA로 transfected 수 웰스의 숫자에 따라 비례 최대 규모. 또한 transfected하는 각 물론 25 μl OPTI - 멤 별도의 1.5 ML Eppendorf 튜브 0.4 μl Lipofectamine 2000 (Invitrogen, 11668-019)에 희석. transfected 수 웰스의 총 개수에 따라 최대이 크기. </li><li> 간단히 와동 희석 cDNA와 Lipofectamine 2000을주고 약 5 분 실온에서 앉아 보자. </li><li> 희석 cDNA, 소용돌이가 들어있는 튜브에 희석 Lipofectamine 2000의 동일한 볼륨을 전송하고 혼합물이 15 분 ~ 2 시간을 위해 실내 온도에 앉아 보자. 이 기간 동안, 49 μl에 / 잘 폴리 - L - ornithine 코팅 판에 Lipofectamine / cDNA 2,000 혼합물을 전송합니다. </li><li> HEK293 세포를 (단계 1.1) Trypsinize, hemocytometer 또는 카운테스 자동 세포 계수기 (Invitrogen, C10227)를 사용하여 셀 밀도를 계산하고, 15 ML 멸균에 (일반적으로 약 ~ 125,000-150,000 전지 / 음) 세포를 원하는 금액을 송금 원뿔 바닥 원심 분리기 튜브. 5 분 1,000 X의 RPM에서 원심 분리기. 10% 열 inactivated 태아 소 혈청과 보충 DMEM 문화 매체 ~ 1,250,00-1,500,000 세포 / ML,에서하지만, 항생제없이 Resuspend 세포. </li><li> 멀티채널 pipetter 또는 MicroFill의 Microplate 디스펜서 (생물 테크)를 사용하여 cDNA - Lipofectamine 2,000 혼합을 포함하고있는 각 물론 98 μl 세포 현탁액을 분배. 접시가 humidified 세포 배양기 문화에 배치하기 전에 최소한 30 분 후드에 앉아 보자. ° C, 5 % CO 2 20-44 시간을위한 37 알을 품다. </li></ol> 안정 TRPA1을 표현하는 HEK293 세포에 대한 <ol start="8"><li> 코트 96 - 웰 플레이트 단계 1.2를 따르십시오. </li><li> inducible HEK293 - hTRPA1 셀 라인은 넉넉한 (Invitrogen, 박사 A. Patapoutian (스크립스 연구소)에 의해 제공하고 야생 타입 HEK293 세포 (단계 1.1)과 같은 매체를 유지하지만, 5 μg / ML blasticidin로 보충했습니다 A1113902) 50 μg / ML hygromycin B (Invitrogen, 10687010). 세포 밀도가 약 90 %에 도달하면, 세포를 trypsinize, 세포 밀도를 계산하고, 5 분 1000 X RPM에서 세포의 원하는 금액을 원심 분리기. doxycycline (피셔, BP265​​35, 0.25 μg / ML)로 보충 같은 문화 매체 ~ 1,000,000 세포 / ML의 밀도에 Resuspend 세포. </li><li> μl / 잘 멀티채널 pipetter 또는 MicroFill (바이오 테크)를 사용하여 100 폴리 - L - ornithine 코팅 접시에 세포 현탁액을 분배. 접시가 humidified 세포 배양기 문화에 배치하기 전에 최소한 30 분 후드에 앉아 보자. 37 품어 ° C 5 % CO 16 2-24 시간. </li></ol> 2. 솔루션 <ol><li> 행크의 버퍼 소금 용액 (HBSS)은 세포 솔루션으로 사용됩니다. 그것은 (MM)를 포함 137 NaCl, 5.4 KCl, 0.25 나 2 HPO 4, 0.44을 KH 2 PO 4, 1.3 CaCl 2, 1.0 MgSO 4, 1.0 1N NaOH를 사용하여 7.4으로 조정 산도와 MgCl 2, 10 포도당, 10 HEPES). </li><li> Fluo - 4 검정 버퍼 HBSS에 probenecid이 MM이 포함되어 있습니다. 0.5 N NaOH 250 MM 주식 probenecid (시그마, P8761)을 확인합니다. 1:125의 비율로 희석 HBSS. 1 N HCL을 사용하여 산도에 7.4을 재조 정해. </li></ol> 3. Fluo - 4이라서 셀 로딩 <ol><li> 1 ㎜의 주식 솔루션을 만들기 위해 912 μl 무수 DMSO 1 MG Fluo - 4시 (Invitrogen, F14202)를 디졸브. Fluo - 4 재고 솔루션을 적어도 한 달 동안 -20 ° C에 저장할 수 있습니다 AM. </li><li> 10 μl 1 ㎜를 섞어 Fluo - 4 다음 10 μl Pluronic F - 127 (Invitrogen, P3000MP, DMSO의 20 % (W / V) 솔루션) 5 ML Fluo - 4 분석 완충액 (단계 2.2)로 전체 내용을 전달 AM (시그마, A9418) 0.1 % 소 혈청 알부민과 보완. </li><li> microplate 와셔 (예 : 바이오 테크에서 ELx405TM 등)를 사용하여 80 μl / 음 2 번에서 HBSS와 (단계 1.7 또는 1.10) 96 - 웰 플레이트에 성장 세포를 씻으십시오. </li><li> Fluo 추가-4는 μl / 잘 다채널 pipetter 또는 MicroFill을 (바이오 테크)를 사용하여 37에서 번호판을 품어 50 (3.2 단계) 솔루션을 로딩 AM ° C 1 시간을 위해. </li><li> ELx405TM의 microplate 와셔를 사용하여 Fluo - 4 분석 완충액 (단계 2.2.)으로 세포에게 3 번 씻으십시오. 마지막 세척 후, 각 우물에 Fluo - 4 검정 버퍼의 80 μl를 떠나. </li></ol> 4. 복합 접시의 준비 <ol><li> 1 시간 셀 로딩 기간 동안, Fluo - 4 검정 버퍼에 원하는 최종 농도의 3 배의 agonists의 3 배 dilutions (또는 antagonists)의 시리즈를 준비합니다. </li><li> 96 - 웰 복합 판으로 희석 복합 솔루션의 피펫 250-300 μl. FlexStation 3 한 번에 하나의 컬럼을 읽고 있기 때문에 그것이 가능한 때마다 복합 판의 동일한 열에있는 화합물과 해당 긍정적이고 부정적인 컨트롤의 시리얼 dilutions을 가지고 배열. </li></ol> 5. FlexStation 3 플레이트 읽기 <ol><li> (FlexStation 3, 컴퓨터, 그리고 SoftMax 프로 5.2 소프트웨어) 시스템을 켜십시오 </li><li> 새 파일 이름으로 실험을 저장합니다. 설정에서 플렉스 모드를 선택하고 다음 값을 입력합니다 : </li></ol> 프로토콜 # 1 작용제 효과 <img src="/files/ftp_upload/3149/3149table1.jpg" alt="표 1" /> 프로토콜 # 2 길항제 효과 <img src="/files/ftp_upload/3149/3149table2.jpg" alt="표 2" /><ol start="3"><li> FlexStation 3 해당 트레이에 새로운 팁 상자, 복합 판과 독서 접시를 놓고 버튼을 &quot;읽기&quot;를 클릭하십시오. FlexStation 3 한 번에 하나의 컬럼을 읽고 독서를 방해하지 않고 프로그램된 시간 지점에서 화합물을 추가합니다. 그것은 하나의 열로 표시하고 기계가 모든 열을 읽기까지로 단계 5.2 사용자에 의해 프로그램 수행 화합물 추가로 다음 칼럼을 읽고 계속을 읽는 끝내고 약 3 분 (프로토콜 # 2 5 분) 정도 소요됩니다. </li></ol> 6. 데이터 분석 <ol><li> 실험 데이터는 직접 SoftMax 프로 5.2 소프트웨어를 사용하거나 복사 및 Microsoft Excel과 같은 스프레드 시트 프로그램, 붙여넣을 처리할 수 있습니다. TRPA1 작용제의 flufenamic 수 소산​​ (FFA)의 농도 - 반응 곡선은 다음과 같은 최소 제곱 피팅 루틴을 사용하여 구성되어 있습니다. <img src="/files/ftp_upload/3149/3149eq1.jpg" alt="방정식 1" /> , Y가 관찰된 반응 어디에, Y 맥스가 표준 최대한의 응답이며, C는 해당 약물 농도이며, EC 50 반 최대한의 반응을 evoked 농도이며, N H는 명백한 힐 계수이다. putative TRPA1 길항제 화합물에 대한 농도 - 저해 곡선은이 FFA (100 μm의)의 고정 농도를 사용하고, 점차적으로이 다음에 맞는 최소 제곱으로 계산됩니다 화합물의 복합 A. IC 50 가치의 농도를 증가하여 얻을 수있다 방정식. <img src="/files/ftp_upload/3149/3149eq2.jpg" alt="방정식 2" /> 어디 Y는 관찰된 반응이고 Y 최대가 최대 응답을 표준인데, [개미]에 해당 화합물의 농도이며, IC 50 FFA - evoked 응답의 절반 최대한 억제를 생산 길항제 농도이며, N H는 분명 힐입니다 계수. </li></ol> 7. 대표 결과 : TRPA1는 +에 민감한 염료 칼슘 2, Fluo - 4 (1-2)에 의해 감지 수있는 칼슘 유입에 이르게 활성화하는 칼슘 2 + - 투과 양이온 채널이다. HEK293 - hTRPA1 안정적인 세포 라인 TRPA1 agonists과 antagonists의 농도 - 반응 관계를 연구하는 데 사용되었다. 전지, Fluo - 4이라서 로드된 분석 버퍼의 80 μl에 배치하고, FlexStation 3을 사용 읽어되었습니다. 30 s에 대한 기준 형광을 기록 후, TRPA1 작용제, FFA의 시리얼 희석 40 μl, 원하는 최종 농도의 3 배 각은 fluidics 모듈의 일부로 포함 멀티 채널 pipetter을 통해 세포에 추가되었습니다 FlexStation 3. 형광 변경 사항은 추가로 180 S.에 대한 감시했다 각각 F와 F 0 관심과 독서의 시작 시점에 형광 값이다 - (F 0 F), 각 잘 대해 상대적 Fluo - 4 형광 변경 ΔF =으로 표현했다. FFA 농도의 일련의에 대한 응답으로 형광 변화 시간 코스의 대표 성분은 그림에 표시됩니다. 1. 최대한 응답 수준은 유사하지만, 활성화 반응 속도론은 낮은 FFA의 농도보다 더 높은에서 명확하게 빠른이며, 마지막 네 FFA의 농도에 대한 참고. 따라서 이러한 차이, 농도를 구별 - 반응 관계는 FFA의 다양한 농도 (그림 2)뿐만 아니라시 형광 증가의 초기 속도를 (경사) 측정에 의해 건설되었습니다. 하프 최대한의 효과FFA의 이브 농도 (EC 50) 55.4 μm의 (N = 3)으로 결정되었다. putative TRPA1 길항제를 테스트하려면 A를 화합물, 우리는 프로토콜 # 2 (2 단계)을 사용하여 비슷한 실험을 수행했습니다. 이 경우에는 길항제의 dilutions 일련의 30 S (배 원하는 최종 농도 40 μl)에 추가된 및 FFA는 (100 μm의의 최종 농도 300 μm의 60 μl) 나중에 180의 추가되었습니다. 컴파 운드 선량 - dependently 4.3 μm의의 반 최대한 억제 농도 (IC 50) (N = 3) (그림 2)와 FFA에 TRPA1 세포의 응답을 감소. <img src="/files/ftp_upload/3149/3149fig1.jpg" alt="그림 1"/> FFA에 의해 인간 TRPA1의 그림 1. 농도 - 의존 활성화. 성분은 시간이 지남에 따라 형광의 FFA - evoked 증가 (처음 60 S에서만 응답이 더 FFA - evoked 응답의 빠른 반응 속도론을 설명하는 표시됩니다)입니다. 높은 농도의 FFA 함께 형광 증가 evoked 점에 유의하십시오 훨씬 빠른 운동 낮은 농도에서보다 정상 형광 수준과 유사했다하더라도. <img src="/files/ftp_upload/3149/3149fig2.jpg" alt="그림 2"/> 그림 2 농도 -. FFA - 유도 형광 변화와 화합물 안정 HEK293 세포에 표현 인간 TRPA1에 FFA 반응의 유도 억제를위한 대응 관계. Δ, 집중력 - FFA의 반응 관계가 FFA 농도의 변화를 추가시 표준 형광 증가의 초기 속도를 (경사) 측정에 의해 결정되었다. O, putative TRPA1 길항제에 의해 FFA - evoked 칼슘 반응의 억제의 농도 - 반응 관계, 화합물 A.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+concentrations+during+"chemical+hypoxia"+and+excitotoxic+neuronal+injury.">Intracellular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury.</a> J Neurosci. 11, 2545-2551 (1991).</li><li>George, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+channel+activation+augments+NMDA+receptor+function+and+promotes+neurite+outgrowth+in+immature+cerebrocortical+neurons.">Sodium channel activation augments NMDA receptor function and promotes neurite outgrowth in immature cerebrocortical neurons.</a> J Neurosci. 29, 3288-3301 (2009).</li><li>Price, K. L., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FlexStation+examination+of+5-HT3+receptor+function+using+Ca2++-+and+membrane+potential-sensitive+dyes:+advantages+and+potential+problems.">FlexStation examination of 5-HT3 receptor function using Ca2+ - and membrane potential-sensitive dyes: advantages and potential problems.</a> J Neurosci Methods. 149, 172-177 (2005).</li><li>Laude, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+functional+assays+of+recombinant+IP3+receptors.">Rapid functional assays of recombinant IP3 receptors.</a> Cell Calcium. 38, 45-51 (2005).</li><li>Marincsak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analgesic+drug,+tramadol,+acts+as+an+agonist+of+the+transient+receptor+potential+vanilloid-1.">The analgesic drug, tramadol, acts as an agonist of the transient receptor potential vanilloid-1.</a> Anesth Analg. 106, 1890-1896 (2008).</li><li>Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+high+throughput+screening+assays+against+three+subtypes+of+Ca(v)3+T-type+channels+using+molecular+and+pharmacologic+approaches.+Assay+Drug+Dev+Technol.">Validation of high throughput screening assays against three subtypes of Ca(v)3 T-type channels using molecular and pharmacologic approaches. Assay Drug Dev Technol.</a> 5, 191-203 (2007).</li><li>Christensen, B. N., Kochukov, M., McNearney, T. A., Taglialatela, G., Westlund, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton-sensing+G+protein-coupled+receptor+mobilizes+calcium+in+human+synovial+cells.">Proton-sensing G protein-coupled receptor mobilizes calcium in human synovial cells.</a> Am J Physiol Cell Physiol. 289, C601-C608 (2005).</li><li>Boulay, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+expression+of+a+novel+mammalian+homolog+of+Drosophila+transient+receptor+potential+(Trp)+involved+in+calcium+entry+secondary+to+activation+of+receptors+coupled+by+the+Gq+class+of+G+protein.">Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein.</a> J Biol Chem. 272, 29672-29680 (1997).</li><li>Nanda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+screening+of+adrenergic+receptors+by+measuring+intracellular+calcium+using+the+FlexStation+scanning+fluorimeter.">Functional screening of adrenergic receptors by measuring intracellular calcium using the FlexStation scanning fluorimeter.</a> Biotechnol J. 4, 417-422 (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

세포내 칼슘 2 +과 같은 신경 전달 물질 릴리즈 (3), 심장 활동 전위 (4), 근육 세포 수축 (5), 세포 신호 전달 (6-7) 및 excitotoxic 세포 죽음 (많은 생리와 병리 학적 조건에서 중요한 역할을 담당 8). 세포내 칼슘 2 + 수준의 측정 기능 + - 투과 채널 플라즈마 막이나 세포 organelles에서 칼슘 2의 변화와 공헌을 명료하게하다하는 데 도움이 위의 프로세스 (9-13)에 CA 2 +...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> FlexStation 3 </td><td> 분자 장치 </td><td> FLEX3 </td><td></td></tr><tr><td> 96 - 음, FlexStation의 Pipet 도움말 </td><td> 분자 장치 </td><td> 9000-0912 </td><td></td></tr><tr><td> 96 - 웰 플레이트 </td><td> 그라 이너 바이오 - 원 </td><td> 655090 </td><td></td></tr><tr><td> 96 - 웰 지우기 플랫 바텀 플레이트 </td><td> 코스타 </td><td> 3595 </td><td></td></tr><tr><td> DMEM </td><td> Invitrogen </td><td> 12430-104 </td><td></td></tr><tr><td> Opti - 멤 </td><td> Invitrogen </td><td> 31985-070 </td><td></td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668-019 </td><td></td></tr><tr><td> AM Fluo - 4 </td><td> Invitrogen </td><td> F14202 </td><td></td></tr><tr><td> PluronicF - 127 </td><td> Invitrogen </td><td> P3000MP </td><td></td></tr><tr><td> 폴리 - L - ornithine </td><td> 시그마 </td><td> P36551 </td><td></td></tr><tr><td> probenecid </td><td> 시그마 </td><td> P8761 </td><td></td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 </td><td> A9418 </td><td></td></tr></tbody></table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

분자 장치 &#39;FlexStation 3 멀티 잘 접시에 자동 형광 측정 가능한 benchtop 다중 모드 microplate 리더입니다. 그것은 학술 설정에서 중간에 높은 처리량 화면에 이상적입니다. 그것은 멀티 채널 pipetter과 기계가 형광 시약의 다양한 형광 변화를 모니터링하기 위해 한 번에 한 컬럼을 읽고 갖춘 통합 유체 전송 모듈이 있습니다. 예를 들어, FlexStation 3 + - 투과 이온 채널과 G 단백질 결합 수용체 세포 무료 칼슘 2 + 수준의 변화를 측정하여 칼슘 2의 기능을 연구하는 데 사용되었습니다. 과도 수용체 잠재적인 (TRP) 채널은 많은 생리와 pathophysiological 기능에 중요한 역할을 nonselective 양이온 채널의 큰 가족입니다. TRP 채널의 대부분은 칼슘 투과하고 활성화시 칼슘 유입을 유도. 이 비디오에서는, 우리는 TRPA1 채널, 다양한 유해 자극에 대해 분자 센서의 약리 프로필을 공부 FlexStation 3 응용 프로그램을 보여줍니다. transiently 또는 안정 96 - 웰 플레이트에서 자란 인간 TRPA1 채널을, 표현 HEK293 세포가 + 민감한 형광 염료 칼슘 2, Fluo - 4, 이러한 세포의 실시간 형광 변경 로드된은의 적용 전과하는 동안 측정 FlexStation 3 플렉스 모드를 사용하여 TRPA1 아고 니스트. putative TRPA1 길항제의 영향도 조사했다. 데이터는 TRPA1 activators와 억제제의 농도 - 반응 관계를 구축하기 위해 SoftMax Pro 소프트웨어에서 전송됩니다.

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

이 동영상은 FlexStation 3을 사용 인간 TRPA1 채널의 약리 프로필을 공부에 대한 자세한 프로토콜을 제공합니다. 프로토콜은 세포 준비, 염료 로딩과 microplate 리더 FlexStation 3 작업의 세부 사항을 다룹니다.

FlexStation 3을 사용 TRP 채널 기능의 높은 처리량 검사하는 매체를위한 셀 기반의 칼슘 분석

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

cell-based-calcium-assay-for-medium-to-high-throughput-screening-trp

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

21.0K 조회수.  The University of Texas Health Science Center at Houston. 이 동영상은 FlexStation 3을 사용 인간 TRPA1 채널의 약리 프로필을 공부에 대한 자세한 프로토콜을 제공합니다. 프로토콜은 세포 준비, 염료 로딩과 microplate 리더 FlexStation 3 작업의 세부 사항을 다룹니다.

비디오: Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

Biomaterials 높은 처리량 발견을위한 폴리머 Microarrays

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

연구

생체공학

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

높은 처리량 Antifungal 약물 검사를위한 칸디다 albicans Biofilm 칩 (CA BChip)

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

마이크로 유체 플랫폼을 사용하여 높은 처리량 단백질 표현 생성기

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 &#176;C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.

Here we present a protocol to synthesize novel, high-aspect ratio biocomposites under biological conditions and in liquid media. The biocomposites scale from nanometers to micrometers in diameter and length, respectively. Copper nanoparticles (CNPs) and copper sulfate combined with cystine are the key components for the synthesis.

생물 액체 배지에서 확장 성, 금속 높은 가로 세로 비율 나노 복합 재료의 생성

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper ...

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Tomoauto 사용 : 프로토콜 높은 처리량 자동화 된 곳을 알아내는 - 전자 단층 촬영을 위해

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

의료 기기에 대한 박테리아의 높은 처리량 식별 구충제 폴리머

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

주 사용 재생 치료와 높은 처리량 마약 검사에 대 한 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

중국 햄스터 난소 세포 재배에서 고처리량 자동화 마이크로 바이오리액터를 사용한 공정 최적화

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

슈도모나스 주사기 pv에 대한 저항의 높은 처리량 식별. 모종 홍수 분석을 사용하여 토마토토마토

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

고처리량 미세 유체 장치를 사용하여 동적 환경 조건의 생성

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...