우리는 그녀의 친절한 도움 필립 워렌 감사하고 싶습니다.

1. 경계 분석 : <ol><li> 기본 준비 : 상공 회의소 슬라이드는 폴리 - D - 라이신 야간 및 멸균 유리 슬라이드로 코팅 아르는 실험을 시작하기 전에 준비가되어 있습니다. 매체는 Dulbecco의 수정된 이글의 10% 소태아 혈청 (FCS)와 보충 중간 (DMEM),, 1 % 페니실린 - 스트렙토 마이신 - Fungizon (PSF)를 사용하여 준비가되어 있습니다. 또한 칼슘 / 마그네슘 무료 행크스 균형 소금 용액 (HBSS)의 병이 준비됩니다. </li><li> 플라스크에 교양 기본 쥐 Schwann 세포는 0.1 % 트립신을 사용하여 3 분 trypsinized 있습니다. 기본 쥐 astrocyte 문화는 0.1 % 트립신을 사용하여 5 분 trypsinized입니다. 트립신은 DMEM를 추가하면 10% FCS, 1 % PSF로 보충하여 inactived 있습니다. </li><li> Trypsinized Schwann 세포와 astrocytes 15 ML 팰컨 튜브를 별도로 전송하고 5 분 300G에 centrifuged 있습니다. </li><li> Schwann 세포와 astrocytes은 (세포 밀도의 변화에 대한 대표적인 결과 섹션을 참조하십시오) 2 × 10 6 밀도에 다시 중지합니다. </li><li> Schwann 세포 현탁액의 50 μL 방울 PDL 코팅 챔버 슬라이드 우물 중 한 끝에 배치됩니다. 유리 스트립은 바로 가장자리를 생성하기 위해 챔버의 중심으로 얼룩 드롭을하는 데 사용되었다. (유리 커터를 사용하여 직사각형 유리 coverslips 그것은 경계 assays 사용 챔버 슬라이드의 너비에 맞게 것이 길이 방향 있도록 절단됩니다) </li><li> astrocyte 정지 50 μL의 두 번째 드롭 같은 우물의 반대 끝에 위치하고 사이에 작은 0.2mm 간격으로 첫 번째 드롭의 얼룩에 평행 직선 모서리를 만드는 중심으로 발랐습니다. </li><li> 챔버는 방울이 37 인큐베이터에 배치됩니다 ° C를 2 시간 포함하는 슬라이드. </li><li> 2 시간 후에, 문화가 아닌 연결된 세포 파편과 DMEM / 10 % PSF가 추가 FCS / 1 %를 포함하는 새로운 매체를 제거하는 DMEM로 씻어되었습니다. 참고 : DMEM / 10 % FCS / PSF가 forskolin (2μM)와 보빈 뇌하수체 추출 (BPE) (10μg / ML) Schwann 세포의 증식을 자극과 보충 수 있습니다 1퍼센트. BPE는 장기 Schwann 세포 배양을 허용하는 데 필요한 성장 요인을 공급하고 forskolin은 확산을 유도하기 위해 Schwann 세포 내에서 순환 아데노신 monophosphate 수준을 올립니다 </li><li> 전지는 37 ° C, 7퍼센트 CO 2 배양기에서 약 8-10일에 대한 양식입니다. 매체는 3 일마다 변경해야합니다. 두 셀 전선 결국 서로 연락하고 날카로운 경계는 두 세포 유형 간의 형태 것입니다. 이 단계에서 실험 경계 형성에 관련된 요인을 분석 수행하실 수 있습니다. </li><li> 공동 문화 4 % 포름 알데히드와 Schwann 세포가 공동 문화의 세포를 식별하는 안티 GFAP 항체와 백신 항체 P75과 astrocytes와 immunostained 수를 사용하여 해결할 수 있습니다. P75 항체는 astrocytes가 부족 Schwann 세포에 낮은 친화 NGF 수용체를 인식합니다. 안티 GFAP 항체는 세포질에 astrocyte glial localizes firbrillary 산성 단백질을 인식합니다. (대표 경계에 대한 그림 1A 참조) </li></ol> 2. 마이 그 레이션 분석 : <ol><li> 네 잘 접시 (15mm 우물)는 PDL 아르 코팅 숙박과 astrocyte monolayers을 만들기위한 준비. </li><li> 기본 astrocyte 문화 0.1 % 트립신을 사용하여 5 분 trypsinized입니다. 트립신은 DMEM 10 % FCS, 1 % PSF 5 분 300G에서 centrifuged 세포와 보충 추가 inactivated입니다. (그림 2 참조) </li><li> Astrocytes는 1x10 5 셀 / ML에서 다시 중지됩니다. 이 솔루션 1 ML 4 잘 접시에 잘 각 PDL 코팅에 추가됩니다. monolayer가 완전히 합류 때까지 Astrocytes는 24~48시간에 대한 양식입니다. </li><li> 병렬 astrocyte monolayers, 원형 PDL - 코팅 유리 coverslips가 50 ML 튜브에 배치와 유리의 작은 조각을 만드는 플라스틱 피펫을 사용하여 조각된다 만드는, astrocyte monolayers에서 Schwann 세포의 마이 그 레이션을 평가합니다. </li><li> 유리 조각은 6 웰 플레이트 접시에 잘으로 전송되며 크기의 조각 적합한 집게를 사용하면 선택 및 기타 우물에 배치됩니다. 5 유리 조각은 6 잘 접시 각 우물에 배치됩니다. </li><li> 플라스크에 교양 기본 Schwann 세포는 0.1 % 트립신을 사용하여 3 분 trypsinized 있습니다. Trypsinized Schwann 세포 15 ML 팔콘 튜브를 분리 전송 5 분 300 G에서 centrifuged 있습니다. Schwann 세포가 DMEM/10 % FCS / 1퍼센트 PSF에서 배 10 6 세포 / ML 다시 중지됩니다. </li><li> Schwann 세포 현탁액 20 μL 방울는 6 잘 접시와 세포에 배치 각 1 부 5 coverslip 조각 37 2 시간 ° C, 7 % CO 2에서 incubated 아르 이상 추가됩니다. </li><li> 2 시간 후에, Schwann 세포는 coverslip 조각에 붙어 있으며 DMEM/10 %가 FCS / 1% PSF는 (forskolin과 10μg / BPE의 ML의 2μM과 보충 조각이 완전히 중간 덮여되도록 각 잘 추가됩니다) . </li><li> 유리 조각이 완전히 죄수 때까지 조각을 포함하는 6 자 플레이트는 다음 24-48시간위한 인큐베이터에 배치됩니다Schwann 세포와 함께 유창. </li><li> 일단 조각이 Schwann 세포와 합류 있으며, 각 조각은 신중하게 날카로운 집게를 사용하여 포착하고 astrocyte monolayer에 거꾸로 땅에 얼굴 그리고 무소속의 세포를 제거하고 HBSS에 담근이다. </li><li> 각 자 그러면 FCS / 1% PSF와 Schwann 세포가 24-48 기간 동안 마이 그 레이션하는 허용하는 DMEM/10 % 1 ML으로 덮여있다. 이 기간 동안 이러한 성장 요인 실험 시약은 Schwann 세포의 마이 그 레이션의 수와 거리에 그들의 효과를 평가하기 위해 추가할 수 있습니다. </li><li> 마이 그 레이션 기간에 따라, 유리 파편은 30 분 4 % paraformaldehyde (PFA)를 추가하여 각 우물의 바닥에 고정하고 있습니다. 다음 고정은 p75에 대한 Schwann 세포 (낮은 친화 NGF 수용체를 인식) 다음 ABC strepavidin 키트 - diaminobenzidine (DAB)를 사용하여 얼룩 빛 현미경 Schwann 세포의 시각화를 허용하려면 immunostain. </li></ol> 3. 대표 결과 : 80-10일 다음 Schwann - astrocyte 공동 문화는 두 세포 유형 간의 날카로운 경계를 표시합니다. Schwann 세포의 증식이 향상으로 forskolin과 BPE는 문화에 사용되는 경우에는이 경계가 더 발음됩니다. 아무런 경계가 10 일 기간 동안 관찰되지 않은 경우 경계가 설립되기 전까지는, 문화의 시간은 더욱 증가 수 있습니다. 세포 밀도의 비율을 변경하면 경계 형성을 증가하는 대안적인 방법입니다. 3시 1분 Schwann의 비율 : 셀 suspensions 8,18를 준비할 때 astrocyte 세포 밀도를 사용할 수 있습니다. 경계가 설립 어디에 선을 그림이 그려진 수 있으며 astrocyte 지역으로 마이 그 레이션 Schwann 세포의 개수가 형광 현미경으로 계산 수 있습니다 intermingling 세포를 평가하기 위해, Schwann 세포와 astrocytes를 식별하는 immunostaining에 따라. 아래 이미지의 두 예제 획득 경계, Schwann 세포와 astrocytes (그림 1A)와 세포가 가난한 기술 (그림 1B)로 인해 다른 지역으로 잘 차별하지 않은 하나의 잘 구성된 인종 차별과 하나가 있습니다. <img src="/files/ftp_upload/2214/2214fig2.jpg" alt="그림 1" /> . 그림 1 바운더리 분석 : Schwann - astrocyte 상호 작용. Schwann 세포의 증식을 자극하는 forskolin과 BPE의 면전에서) 십일 공동 문화 분석은 두 가지 세포 유형 (적색 = P75, 녹색 = GFAP) 사이에 날카로운 경계를 보여줍니다. B) 경계가 형성되지 십일 공동 문화 분석과 두 세포 전선이 혼합. (빨간색 = P75, 녹색 = GFAP)의 경계를 준비​​하는 동안 서로 옆에 astrocyte와 Schwann 세포 방울을 배치하면서이 두 방울의 혼합으로 인해되었습니다. <img src="/files/ftp_upload/2214/2214fig1.jpg" alt="그림 2" /> 그림 2. 합류 astrocyte monolayer 대표 이미지 마이 그 레이션 assays는 다른 세포 유형의 표면 위에 한 셀 유형의 움직임을 평가하고 따라서 경계 형성의에서 다른 현상을 평가합니다. 연습이 monolayers에 반전 coverslips을 보장하기 위해 필요한 것은 Schwann 세포 덮인 날카로운 집게로 유리 파편과 astrocyte monolayers 있습니다. 손상으로 이어질하지 않습니다 Schwann 세포, 마이 그 레이션의 최대 거리 coverslip의 가장자리에서 마이 그 레이션하는 세포의 수를 마이 그 레이션을 평가하는 것은 가벼운 현미경으로 측정할 수 있습니다. 아래 astrocyte monolayer의 표면 (그림 3)에서 DAB의 얼룩을 사용하여 P75 항체와 스테인드 Schwann 세포의 이미지입니다. <img src="/files/ftp_upload/2214/2214fig3.jpg" alt="그림 3" /> 거꾸로 coverslip 분석을 사용하여 그림 3. 마이 그 레이션 분석. A) Schwann 세포는 (갈색, p75 항체와 immunostained) coverslip의 가장자리에서 떨어져 마이 그 레이션. 멀리 coverslip에서 셀 마이 그 레이션의 방향 화살표를 가리 킵니다.

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+migration+is+integrin-dependent+and+inhibited+by+astrocyte-produced.">Schwann cell migration is integrin-dependent and inhibited by astrocyte-produced.</a> Glia. , (2010).</li><li>Santos-Silva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF/heparin+differentially+regulates+Schwann+cell+and+olfactory+ensheathing+cell+interactions+with+astrocytes:+a+role+in+astrocytosis.">FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis.</a> J Neurosci. 27, 7154-7167 (2007).</li><li>Afshari, F. T., Kwok, J. C., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-produced+ephrins+inhibit+schwann+cell+migration+via+VAV2+signaling.">Astrocyte-produced ephrins inhibit schwann cell migration via VAV2 signaling.</a> J Neurosci. 30, 4246-4255 (2007).</li></ol>

관심 없음 충돌 선언하지 않습니다.

위에서 설명한 assays은 Schwann - astrocytes 및 astrocytic 환경에서 제한된 Schwann 세포 마이 그 레이션 사이의 경계 형성에 관련된 여러 요인의 역할을 보여주는 여러 연구에 사용되었습니다. 그것이 전략의 개발 최적화하도록하고 이식 다음 Schwann 세포 이식의 통합을 강화하고 이렇게에서 허용 호스트 조직에 그래프트 뒷부분에서 재생의 axons의 출구를 촉진하는 것처럼 이러한 이벤...

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		<div>
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41966-029</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-088</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>PSF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>BPE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13028-014</td>
<td>&nbsp;</td>
<tr>
<td>Forskolin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>344273</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips</td>
<td>&nbsp;</td>
<td>VWR-International</td>
<td>631-0149</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips(rectangle)</td>
<td>&nbsp;</td>
<td>MENZEL-GLASER (Supplied by Thermo Scientifics)</td>
<td>BB022050A1</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nunc-LabTek </td>
<td>177380</td>
<td>&nbsp;</td>
<tr>
<td>4 well plates, 6 well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>&nbsp;</td>
<td>4 well plates 
6 well plates</td>
</tr>
<tr>
<td>rat p75 antibody</td>
<td>&nbsp;</td>
<td>Millipore, Temecula,California</td>
<td>MAB365</td>
<td>&nbsp;</td>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Dako</td>
<td>Z0334</td>
<td>&nbsp;</td>
<tr>
<td>Secondary antibodies (Alexa-conjugated)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004 A-11034</td>
<td>Goat anti mouse 568 
Goat anti Rabbit 488</td>
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<td>Secondary biotinylated </td>
<td>&nbsp;</td>
<td>Vector</td>
<td>&nbsp;</td>
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<td>DAB tablets </td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>D4418</td>
<td>&nbsp;</td>
<tr>
<td>ABC elite kit</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>PK-6100</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps </td>
<td>&nbsp;</td>
<td>FST</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6168</td>
<td>&nbsp;</td>
<tr>
<td>Fluorosave</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>345789</td>
<td>&nbsp;</td>		</tbody>
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analysis of schwann-astrocyte interactions using in vitro assays

Schwann 세포는 척수 부상 다음과 복구 전략에서 일반적으로 사용되는 셀 중 하나입니다. Schwann 세포가 axonal 재생을 지원하고 secreting 성장 요인의 1,2로 돋아 및 유착 분자 3 세포외 기질 분자 4 증진 성장을 제공할 수 있습니다. 또한 그들은 부상 5 사이트에 demyelinated axons를 myelinate. 그렇지만 이식 이어 Schwann 전지 보형물 사이트에서 이주 없어하고 호스트 astrocytes 6,7로 뒤섞다 없어. Schwann 세포와 astrocytes 사이에 날카로운 경계의 형성이 결과, proximally 및 distally 호스트 조직에 그래프트 다시를 종료하려고 성장 axons에 대한 장애물을 만드는. Schwann 세포와 접촉 Astrocytes도 비대를 받아야하고 억제 분자 8-13을 - 규제. 시험 관내 assays에서 모델 Schwann 세포 astrocyte 상호 작용하는 데 사용되었습니다과 세포 동작을 기본 메커니즘을 이해하는 것이 중요했습니다. 시험 관내 assays에서 이들은 공동 문화 두 가지 세포를 분리 전선에만 작은 격차로 다른 지역을 점유 각 세포 유형으로 두 개의 다른 세포를 사용하여 만든 경계 분석을 포함합니다. 세포 분열 및 마이 그 레이션으로 두 세포 면에서 서로 가까이 그리고 마지막으로 충돌하십시오. 이것은 경계에서 분석하는 두 세포 인구의 동작을 수 있습니다. 같은 기술의 또 다른 변화는 문화의 두 세포 인구를 섞어하고 시간이 지남에 따라 두 세포 유형은 Schwann 세포와 분리 함께 여러 Schwann - astrocyte 경계를 만드는 astrocytes 사이에 섬이 함께 clumped하는 것입니다. 두 세포 유형의 상호 작용을 연구에 사용되는 두 번째 분석은 세포의 움직임이 다른 세포 유형 monolayer 14,15의 표면에 추적할 수있는 마이 그 레이션 분석이다. 이 분석은 일반적으로 바뀌 coverslip 분석으로 알려져 있습니다. Schwann 세포는 작은 유리 파편에 교양하고 그들은 astrocyte monolayers의 표면에 얼굴을 거꾸로하고 마이 그 레이션이 coverslip의 가장자리에서 평가됩니다. 두 assays은 세포 배제와 경계 형성에 관련된 기본 메커니즘을 연구의 수단이되었습니다. 분자의 일부는 이러한 기술이 N - Cadherins 15, 콘드로이틴 황산염의 proteoglycans (CSPGs) 16,17, FGF / 헤파린 18 에프 / Ephrins 19 등이 사용 확인. 이 문서는 stepwise 방식으로 경계 분석 및 마이 그 레이션 분석을 설명하고 발생할 수있는 가능한 기술적인 문제를 명료하게하다 예정입니다.

Watch this Scientific Journal Video about Analysis of Schwann-astrocyte Interactions Using In Vitro Assays at JoVE.com

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

이 문서는 stepwise 방식으로 표현하고자하는 일반적으로 사용되는<em&gt; 체외에서</emSchwann 세포 asrtocyte 상호 작용을 공부에 사용&gt; assays.

사용 Schwann - astrocyte 상호 작용 분석 체외에서 Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

12.2K 조회수.  University of Cambridge. 이 문서는 stepwise 방식으로 표현하고자하는 일반적으로 사용되는 체외에서 assays.

비디오: Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

자기 변조 Biosensing 시스템을 사용하여 생물 학적 Assays의 급속한 동차 검색

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

연구

생물학

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

PDZ-의존 CFTR의 Macromolecular 신호 단지의 체외 분석

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

체외 세포 이동 및 내습 분석 실험에서

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

사전 mRNA의 기판의 3 &#39;끝 분열 : 휴대폰 무료 시스템을 사용하여 RNA 가공 반응 분석<em&gt; 시험관</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

생체 외 분석에서 집계 Hyperphosphorylated 타우 단백질을 사용하여

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

SPRI, 나노 SPRI 및 ELISA 분석 실험을 사용하여 혈청에있는 인간 성장 호르몬의 비교 분석

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

Explanted 망막 조직 세포 외 유출 분석을 사용 하 여 신진 대사 에너지의 측정

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

효 모에 Polyglutamine 독성 분석 실험을 사용 하 여 퓨전 파트너에 형광 단백질의 영향

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

2단계 PCR 및 차세대 16S rRNA 유전자 시퀀싱을 사용한 미생물 분석

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...