우리가 그들의 협력 임상 병리학, 비엔나, 오스트리아의 대학의 연구소에서 교수 Dontscho Kerjaschki, 코리나 Mayrhofer 및 시거드 크레 이거 감사합니다.

세포 배양 <ol><li> GlutaMAX 나는 10 % 태아 송아지 혈청, 50 U / ML 페니실린, 50 μg / ML의 스트렙토 마이신 황산 (Invitrogen)를 포함과 함께 F - 12 햄 매체의 표준 세포 배양 절차를 사용하여 중국어 햄스터 난소 세포를 (CHO - K1) 성장. </li><li> 혈청이없는 미디어로 문화 매체를 교환. 라벨 세포 표면 단백질은 CyDye DIGE 플루어 Cy3 또는 Cy5 최소한의 염료 (아래 섹션 세포 표면 라벨을 참조) 다른 시간 지점에 있었다. </li><li> 각각의 시점과 CyDye DIGE 플루어 Cy2와 레이블에서 세포의 풀 같은 숫자. 각각의 2 - D 겔을위한 내부 표준으로 이들을 사용합니다. </li><li> 실험의 대부분은 CHO - K1 세포로 수행할 수 있지만, 마우스 배아 섬유아 세포 (3T3 L1)와 마우스 복수의 림프종 lymphoblasts (EL4)도 (데이터가 표시되지 않음) 사용할 수 있습니다. </li><li> GlutaMAX II와 DMEM 매체 두 후자의 셀 유형을 성장하지만, 그렇지 않으면, CHO - K1 세포에 사용되는 동일한 조건을 사용합니다. </li></ol> 세포 표면 라벨링 <ol><li> 조심스럽게 계산 및 50-10 x106 세포의 aliquots 나누어 아닌 효소 자기편 세포를 분리. 정지 성장 세포 들어, 분리 단계를 생략하십시오. </li><li> 5 분 약 800 XG에있는 세포를 원심 분리기 suspensions. 매체를 포함하는 supernatants를 제거합니다. </li><li> 1 ML 얼음 차가운 행크의 밸런스드 소금 솔루션 (HBSS) 산도 8.5에서 resuspendion하여 알약을 씻으십시오. 4 800 XG ° 2 분 C에서 Centrifuged. </li><li> 뜨는를 제거하고 200 μl 얼음 차가운 라벨 버퍼에있는 세포 펠렛을 (HBSS 산도 8.5, 1 M 우레아) resuspend. </li><li> 어두운 얼음 속에서 20 분 동안 600 pmol CyDye DIGE 플루어 최소한의 염료로 그대로 세포 라벨. </li><li> 라이신 20 μl 10 MM을 추가하여 반응을 끄다. 10 분 알을 품다. </li><li> 4 800 XG에서 원심 분리 ° C 2 분 뒤에 500 μl HBSS의 산도 7.4에 resuspension에 의해 두 번 표면 라벨 세포를 씻으십시오. </li></ol> 세포 용해 및 분류 <ol><li> 150 μl 차가운 용해 완충액 (7 M의 요소, 2 M 티오 우레아, 4 % 챕스, 30 MM 트리스, 5 MM의 마그네슘 아세테이트 산도 8.5)에 표면 라벨 세포를 Lyse. 가끔 vortexing 최소한 1 H 위해 얼음에 둡니다. </li><li> 4 10 000 XG에 lysates을 원심 ° C를 5 분. 새로운 튜브에 뜨는를 전송합니다. 이 예제는 모든 세포 단백질을 포함하는 비 fractionated 예제입니다. </li><li> 병렬, 멤브레인 및 cytosolic 분수 이전 2 - D 겔 전기 영동에로 (막 분획화 키트를 사용하여 피어스), 위에서 다음 분류 셀 알약을 씻으십시오. 멤브레인 분율은 내부와 세포 표면 막 단백질을 포함하고 있습니다. </li><li> 비교, 표준 Ettan DIGE 절차 3에 따라, 그리고 lyse, 레이블, 그리고 세포 마지막으로, 분류. D 퀀트 키트 (GE 헬스케어) - 2를 사용하여 샘​​플의 단백질 농도를 결정합니다. </li></ol> 2 - D 전기 영동 <ol><li> Immobiline의 DryStrip의 젤, Immobiline DryStrip Reswelling 트레이, 하루 450 μl DeStreak의 Rehydration 솔루션 24cm (0.5 %는 IPG 버퍼)를 사용하여 pH를 3 11NL을 (24cm) Rehydrate. </li><li> 매니폴드의 양극 컵로드하여 DryStrip 젤류을 Immobiline하고 지시에 따라 Ettan IPGphor II IEF 시스템을 사용하여 isoelectric 초점 (IEF)을 수행할 수있는 CyDye - 라벨 샘플 (50 μg 총 단백질에 해당)이 적용됩니다. </li><li> IEF 후, 대형 (26 X 20cm) 12.5 %의 polyacrylamide의 젤 (SDS - PAGE) 위에 두 단계와 장소에 부착된를 평형. 0.5 % 아가로 오스와 중첩 (bromophenol 파란색을 포함하는 버퍼를 실행에). 30 분 5 W / 겔에서 Ettan DALTtwelve 대형 수직 시스템을 사용하여 2 - D 전기 영동을 실행한 다음 15 염료 앞까지 W / 젤은 젤 하단에 도달합니다. </li></ol> 이미징 및 데이터 분석 <ol><li> 2 - D 전기 영동을 마친 후, 태풍 9,410 변수 모드 영상기를 사용하여 Cy2, Cy3 또는 Cy5에 대한 젤을 검사합니다. </li><li> DeCyder 2 - D 차동 분석 소프트웨어 사를 사용하여 막 분수, cytosolic 분수, 비 fractionated 샘플에서 현장지도를 비교합니다. </li></ol> 포스트 - 염색법 <ol><li> 이미징 후, 실버 표준 절차에 따라 5 젤을 얼룩. </li></ol> 단백질 식별 <ol><li> , 수확을 성장뿐만 아닌 fractionated 샘플 위에서 설명한 예비 금액 (10 x106 CHO 세포에서 약 1 MG 총 단백질) 세포를 씻어 및 lyse. 대못으로 Cy5 세포 표면 라벨 샘플을 (위 참조)를 사용하고, 단백질의 unlabelled 예비 금액과 함께 적용됩니다. </li><li> 위에서 설명한 2 - D 전기 영동을 수행하지만, 이번에는, aplly 600 μg 레이블이 지정되지 않은 세포는 양극 컵 응용 프로그램에 의해 스파이크 표시 50 μg 세포 표면과 함께 lysate. 올바른 장소 따기 수 있도록 참조 마커를 사용하여 할 곳추천 3에 따라 겔 주조하기 전에 트윈은 유리 접시. </li><li> 딥 퍼플 총 단백질 3 얼룩 사용하는 총 단백질 얼룩 다음 Cy5 세포 표면 현장지도를 얻을 수 Cy5 채널에서 젤 스캔. </li><li> DeCyder 사용하여 두 개의 현장지도를 2 - D 소프트웨어를 함께 매치하고 싸이 오 표시된 세포 표면 단백질에 해당하는 모든 장소에 대한 선택 목록을 만들 수 있습니다. Ettan 명소 처리 스테이션을 사용하여 세포 표면 단백질을 선택, 겔 플러그에서 추출하고, Ettan의 소화조를 사용하여 trypsinate. 든 - TOF 질량 분광법을 사용하여 식별합니다. </li></ol> 표 1. Ettan DIGE 실험은 그림 1에있는 세포 표면 단백질 라벨 프로토콜에 따라 분류 혈청 고갈 세포의 샘플을 사용하여 수행되었습니다. <table style="width: 517px; height: 139px;" border="1" cellspacing="0" cellpadding="0"><tbody><tr><td style="text-align:center; font-weight:bold"> 견본 </td><td style="text-align:center; font-weight:bold"> 혈청 고갈의 시간 </td><td style="text-align:center; font-weight:bold"> CyDye과 Labelled </td><td style="text-align:center; font-weight:bold"> 젤 번호 </td></tr><tr><td style="text-align:center;"> 1 </td><td style="text-align:center;"> - </td><td style="text-align:center;"> 싸이 3, 싸이 2 </td><td style="text-align:center;"> 1 </td></tr><tr><td style="text-align:center;"> 2 </td><td style="text-align:center;"> 삼십분 </td><td style="text-align:center;"> 싸이 5, 싸이 2 </td><td style="text-align:center;"> 1 </td></tr><tr><td style="text-align:center;"> 3 </td><td style="text-align:center;"> 이시간 </td><td style="text-align:center;"> 싸이 3, 싸이 2 </td><td style="text-align:center;"> 2 </td></tr><tr><td style="text-align:center;"> 4 </td><td style="text-align:center;"> 사시간 </td><td style="text-align:center;"> 싸이 5, 싸이 2 </td><td style="text-align:center;"> 2 </td></tr><tr><td style="text-align:center;"> 5 </td><td style="text-align:center;"> 16시간 </td><td style="text-align:center;"> 싸이 5, 싸이 2 </td><td style="text-align:center;"> 3 </td></tr></tbody></table>

<ol>
	<li>Jang, J. H., Hanash, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+of+the+cell+surface+proteome.">Profiling of the cell surface proteome.</a> Proteomics. 3, 1947-1954 (2003).</li><li>Shin, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+profiling+of+the+cell+surface+proteome+of+cancer+cells+uncovers+an+abundance+of+proteins+with+chaperone+function.">Global profiling of the cell surface proteome of cancer cells uncovers an abundance of proteins with chaperone function.</a> J Biol Chem. 9, 7607-7616 (2003).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ettan+DIGE+System+User+Manual.">Ettan DIGE System User Manual.</a> GE Healthcare user manual 18-1164-40. , (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DeCyder+2-D+Differential+Analysis+Software+v+6.0.+GE+Healthcare+data+file+11-0011-98+Edition+AA.">DeCyder 2-D Differential Analysis Software v 6.0. GE Healthcare data file 11-0011-98 Edition AA.</a> , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-D+Electrophoresis+using+immobilized+pH+gradients,+principles+and+methods.">2-D Electrophoresis using immobilized pH gradients, principles and methods.</a> GE Healthcare handbook 80-6429-60 Edition AB. , (2002).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CyDye+DIGE+fluors+and+labeling+kits.+GE+Healthcare+data+file+18-1164-84+Edition+AB.">CyDye DIGE fluors and labeling kits. GE Healthcare data file 18-1164-84 Edition AB.</a> , (2003).</li><li>Mayrhofer, C., Krieger, S., Allmaier, G., Kerjaschki, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DIGE+compatible+labeling+of+surface+proteins+on+vital+cells+in+vitro+and+in+vivo.">DIGE compatible labeling of surface proteins on vital cells in vitro and in vivo.</a> Proteomics. 2, 579-5785 (2006).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+labeling+of+cell-surface+proteins+using+CyDye+DIGE+Fluor+minimal+dyes.+GE+Healthcare+Application+Note+11-0033-92+Edition+AB.">Selective labeling of cell-surface proteins using CyDye DIGE Fluor minimal dyes. GE Healthcare Application Note 11-0033-92 Edition AB.</a> , (2005).</li></ol>

단백질 농도 두 상표 워크플로우의 개요는 그림 1에 표시됩니다. 세포 표면 단백질 라벨 프로토콜에 따라 분류하면 세포가 그대로 있기 때문에, 단지 세포 표면 단백질이 염색에 노출되어 있습니다. 세포 내부뿐만 아니라 외부 라벨 부착 및 단백질은 (그림 1) 표시되기 전에 표준 Ettan DIGE 프로토콜에서는, 세포가 lysed 있습니다. 세포 - 표면 단백질이 특별히 quantitated 수 없으므?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CyDye DIGE Kit, 2 nmol</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>28-9345-30</td>
<td>&nbsp;</td>
<tr>
<td>2-D Quant Kit</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>80-6484-51</td>
<td>&nbsp;</td>
<tr>
<td>IPG Buffer pH 3-11NL</td>
<td>Reagent</td>
<td>GE Heallthcare</td>
<td>17-6004-40</td>
<td>&nbsp;</td>
<tr>
<td>Immobiline DryStrip pH 3-11NL, 24 cm</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-6003-77</td>
<td>&nbsp;</td>
<tr>
<td>IPGbox</td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>28-9334-65</td>
<td>&nbsp;</td>
<tr>
<td>IPGbox kit</td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>28-9334-92</td>
<td>&nbsp;</td>
<tr>
<td>DeStreak Rehydration solution</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-6003-19</td>
<td>&nbsp;</td>
<tr>
<td>Immobiline DryStrip Cover Fluid</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1335-01</td>
<td>&nbsp;</td>
<tr>
<td>Ettan IPGphor 3 IEF Unit</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>11-0033-64</td>
<td>&nbsp;</td>
<tr>
<td>Ettan IPGphor Manifold</td>
<td>Instrument component</td>
<td>GE Healthcare</td>
<td>80-6498-38</td>
<td>&nbsp;</td>
<tr>
<td>Ettan DALTtwelve Gel Caster </td>
<td>Tool</td>
<td>GE Healthcare</td>
<td>80-6467-22</td>
<td>&nbsp;</td>
<tr>
<td>Ettan DALTtwelve Separation Unit and Power Supply/Control Unit</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>80-6466-46</td>
<td>230 V unit: 80-6466-27</td>
</tr>
<tr>
<td>Typhoon 9410 Variable Mode Imager</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>63-0055-81</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Spot Picker</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1145-28</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Digester</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-68</td>
<td>&nbsp;</td>
<tr>
<td>Ettan Spotter</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-67</td>
<td>&nbsp;</td>
<tr>
<td>Ettan MALDI-TOF</td>
<td>Instrument</td>
<td>GE Healthcare</td>
<td>18-1142-33</td>
<td>&nbsp;</td>
<tr>
<td>DeCyder 2-D Differential Analysis Software</td>
<td>Other</td>
<td>GE Healthcare</td>
<td>28-4012-01</td>
<td>&nbsp;</td>
<tr>
<td>ImageQuant Analysis Software</td>
<td>Other</td>
<td>GE Healthcare</td>
<td>28-9236-62</td>
<td>&nbsp;</td>
<tr>
<td>Urea</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1319-01</td>
<td>&nbsp;</td>
<tr>
<td>CHAPS</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1314-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Dithiothreitol (DTT)</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1318-01</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol Blue</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1329-01</td>
<td>&nbsp;</td>
<tr>
<td>Tris</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1321-01</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Dodecylsulfate (SDS)</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1313-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Glycerol 87%</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1325-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne ReadySol IEF 40% T, 3% C</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1310-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne TEMED</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1312-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Ammonium Persulfate</td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1311-01</td>
<td>&nbsp;</td>
<tr>
<td>PlusOne Glycine </td>
<td>Reagent</td>
<td>GE Healthcare</td>
<td>17-1323-01</td>
<td>&nbsp;</td>
<tr>
<td>2-D Sample Prep for membrane proteins</td>
<td>Reagent</td>
<td>Pierce</td>
<td>89864</td>
<td>&nbsp;</td>
<tr>
<td>Streptomycin sulphate</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

selective labelling of cell-surface proteins using cydye dige fluor minimal dyes

표면 단백질은 환경에 반응하고 이웃 세포와 상호 작용하는 세포의 능력의 핵심입니다. 그들은 거의 모든 세포 신호의 inducers 것으로 알려져 있습니다. 또한, 그들은 환경 적응 및 약물 치료에 중요한 역할을하고, 종종 질병 pathogenesis 및 병리 (1)에 참여하고 있습니다. 단백질 - 단백질 상호 작용은 신호 경로에 내장하고 있으며, 이러한 복잡한 생물 학적 과정에 더 많은 통찰력을 얻을 수, 민감하고 안정적​​인 방법은 세포 표면 단백질을 연구 필요합니다. 2 차원 (2 - D) 전기가 차등 변경 사항을 공부 복잡한 단백질 샘플에 생체와 다른 대상의 검출을 위해 광범위하게 사용됩니다. 그들의 낮은 풍부 (세포 단백질의 12 %)로 인해 일부 세포 표면 단백질은 분류 또는 일부 다른 유형의 이득이없는 2 - D 겔에서 감지하기가 어렵습니다. 그들은 또한 종종 제대로 자신의 소수성 자연과 높은 분자량 (2)에 의한 2 - D의 젤으로 표시됩니다. 본 연구에서는, 우리는 단백질이 중요한 그룹의 특정 라벨 및 감지를위한 CyDye DIGE 플루어 최소한의 염료를 사용하여 그대로 전지에 대한 새로운 프로토콜을 제시한다. 결과는 세포 내 단백질의 최소한의 라벨과 함께 세포 표면 단백질의 다수의 특정 라벨을 보여주었다. 이 프로토콜은 사용하는 빠른 속도, 간단하고, 세 CyDye DIGE 플루어 최소한의 염료는 (싸이 2, 싸이 3 싸이 5) 세포 - 표면 단백질에 라벨을 사용할 수 있습니다. 이러한 기능은 DeCyder 2 - D 차동 분석 소프트웨어를 사용하여 단백질 발현 변화 Ettan DIGE 기술과 분석 2 - D 형광 차이점 젤 전기 영동 (2 - D DIGE)를 사용하여 멀티플렉싱을위한 수 있습니다. 세포 - 표면 단백질의 수준은 시간을 다양한 길이에 대한 CHO 세포를 (표 1 참조)의 혈청 기아 동안 미행했다. 풍부의 작은 변화는 높은 정밀도로 감지했고, 결과는 정의 통계적 방법으로 지원됩니다.

Watch this Scientific Journal Video about Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes at JoVE.com

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

단순하고 구체적인 방법은 분획화 단계없이 형광 라벨 부착 및 세포 표면 단백질의 향상된 감지를위한 시연했다. 세포 표면 단백질의 차동 풍부한는 (2 - D) 2 차원 전기 영동과 Ettan ™ DIGE 기술을 사용하여 분석했다.

CyDye DIGE 플루어 최소 염료를 사용하여 셀 표면 단백질의 선택적 Labelling

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

selective-labelling-cell-surface-proteins-using-cydye-dige-fluor

Research

JoVE Journal

Biology

15.1K 조회수.  GE Healthcare Bio-Sciences AB. 단순하고 구체적인 방법은 분획화 단계없이 형광 라벨 부착 및 세포 표면 단백질의 향상된 감지를위한 시연했다. 세포 표면 단백질의 차동 풍부한는 (2 - D) 2 차원 전기 영동과 Ettan ™ DIGE 기술을 사용하여 분석했다.

비디오: Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

전압에 민감한 염료를 사용하여 기니 돼지 장용 네트워크에서 전기 활동의 광학 녹음

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

연구

생물학

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

낮은 친화력 세포 수용체 - 리간드 상호 작용의 확장 가능한 감지를위한 욕망 기반의 세포 상호 작용 검사 (AVEXIS)

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of different cell types in vitro and in vivo.1-5 Although there are literally thousands of publications using such dyes, some of the most commonly encountered cell tracking applications include monitoring of:
<ol>
	<li>stem and progenitor cell quiescence, proliferation and/or differentiation6-8</li>
	<li>antigen-driven membrane transfer9 and/or precursor cell proliferation3,4,10-18 and</li>
	<li>immune regulatory and effector cell function1,18-21.</li>
</ol>
Commercially available cell tracking dyes vary widely in their chemistries and fluorescence properties but the great majority fall into one of two classes based on their mechanism of cell labeling. "Membrane dyes", typified by PKH26, are highly lipophilic dyes that partition stably but non-covalently into cell membranes1,2,11. "Protein dyes", typified by CFSE, are amino-reactive dyes that form stable covalent bonds with cell proteins4,16,18. Each class has its own advantages and limitations. The key to their successful use, particularly in multicolor studies where multiple dyes are used to track different cell types, is therefore to understand the critical issues enabling optimal use of each class2-4,16,18,24.
The protocols included here highlight three common causes of poor or variable results when using cell-tracking dyes. These are:
<ol>
	<li>Failure to achieve bright, uniform, reproducible labeling. This is a necessary starting point for any cell tracking study but requires attention to different variables when using membrane dyes than when using protein dyes or equilibrium binding reagents such as antibodies.</li>
	<li>Suboptimal fluorochrome combinations and/or failure to include critical compensation controls. Tracking dye fluorescence is typically 102 - 103 times brighter than antibody fluorescence. It is therefore essential to verify that the presence of tracking dye does not compromise the ability to detect other probes being used.</li>
	<li>Failure to obtain a good fit with peak modeling software. Such software allows quantitative comparison of proliferative responses across different populations or stimuli based on precursor frequency or other metrics. Obtaining a good fit, however, requires exclusion of dead/dying cells that can distort dye dilution profiles and matching of the assumptions underlying the model with characteristics of the observed dye dilution profile.</li>
</ol>
Examples given here illustrate how these variables can affect results when using membrane and/or protein dyes to monitor cell proliferation.

Successful use of cell tracking dyes to monitor immune cell function and proliferation involves several critical steps. We describe methods for: 1) obtaining bright, uniform, reproducible label-ing with membrane dyes; 2) selecting fluorochromes and data acquisition conditions; and 3) choosing a model to quantify cell proliferation based on dye dilution.

셀 추적 염료를 사용하여 전지 부문 모니터링에 최적화 된 착색 및 확산 모델링 방법

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

게놈 DNA에서 도보로 5 hydroxymethylcytosine의 선택적 캡처

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

세포 표면 단백질 체학 (Proteomics)에 대한 글리코 펩타이드 캡처

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

Measurement of Extracellular Ion Fluxes Using the Ion-selective Self-referencing Microelectrode Technique

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers such as epithelia also form a barrier that separates the inside from the outside or different compartments of multicellular organisms. A key feature of these barriers is the differential distribution of ions across cell membranes or cell layers. Two properties allow this distribution: 1) membranes and epithelia display selective permeability to specific ions; 2) ions are transported through pumps across cell membranes and cell layers. These properties play crucial roles in maintaining tissue physiology and act as signaling cues after damage, during repair, or under pathological condition. The ion-selective self-referencing microelectrode allows measurements of specific fluxes of ions such as calcium, potassium or sodium at single cell and tissue levels. The microelectrode contains an ionophore cocktail which is selectively permeable to a specific ion. The internal filling solution contains a set concentration of the ion of interest. The electric potential of the microelectrode is determined by the outside concentration of the ion. As the ion concentration varies, the potential of the microelectrode changes as a function of the log of the ion activity. When moved back and forth near a source or sink of the ion (i.e. in a concentration gradient due to ion flux) the microelectrode potential fluctuates at an amplitude proportional to the ion flux/gradient. The amplifier amplifies the microelectrode signal and the output is recorded on computer. The ion flux can then be calculated by Fick&#8217;s law of diffusion using the electrode potential fluctuation, the excursion of microelectrode, and other parameters such as the specific ion mobility. In this paper, we describe in detail the methodology to measure extracellular ion fluxes using the ion-selective self-referencing microelectrode and present some representative results.

Transporters in cell membranes allow differential segregation of ions across cell membranes or cell layers and play crucial roles during tissue physiology, repair and pathology. We describe the ion-selective self-referencing microelectrode that allows the measurement of specific ion fluxes at single cells and tissues in vivo.

이온 선택적 자체 참조 미세 전극 기술을 이용하여 세포 외 이온 플럭스 측정

Cells from animals, plants and single cells are enclosed by a barrier called the cell membrane that separates the cytoplasm from the outside. Cell layers ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

광 안정성 시아닌 염료를 사용하여 파장 변환 DNA 혼성화 프로브의 합성

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Determination of the Relative Cell Surface and Total Expression of Recombinant Ion Channels Using Flow Cytometry

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these multi-spanning transmembrane proteins, with or without change in channel gating, is often postulated to contribute significantly in this process. It has thus become critical to develop a method to quantify the change of the relative cell surface expression of cardiac ion channels on a large scale. Herein, a detailed protocol is provided to determine the relative total and cell surface expression of cardiac L-type calcium channels CaV1.2 and membrane-associated subunits in tsA-201 cells using two-color fluorescent cytometry assays. Compared with other microscopy-based or immunoblotting-based qualitative methods, flow cytometry experiments are fast, reproducible, and large-volume assays that deliver quantifiable end-points on large samples of live cells (ranging from 104 to 106 cells) with similar cellular characteristics in a single flow. Constructs were designed to constitutively express mCherry at the intracellular C-terminus (thus allowing a rapid assessment of the total protein expression) and express an extracellular-facing hemagglutinin (HA) epitope to estimate the cell surface expression of membrane proteins using an anti-HA fluorescence conjugated antibody. To avoid false negative, experiments were also conducted in permeabilized cells to confirm the accessibility and proper expression of the HA epitope. The detailed procedure provides: (1) design of tagged DNA (deoxyribonucleic acid) constructs, (2) lipid-mediated transfection of constructs in tsA-201 cells, (3) culture, harvest, and staining of non-permeabilized and permeabilized cells, and (4) acquisition and analysis of fluorescent signals. Additionally, the basic principles of flow cytometry are explained and the experimental design, including the choice of fluorophores, titration of the HA antibody and control experiments, is thoroughly discussed. This specific approach offers objective relative quantification of the total and cell surface expression of ion channels that can be extended to study ion pumps and plasma membrane transporters.

Inherited cardiac arrhythmias are often caused by mutations that alter the surface delivery of one or more ion channels. Here, we adapt flow cytometry assays to provide a quantification of the relative total and cell surface protein expression of recombinant ion channels expressed in tsA-201 cells.

유동 세포 계측법을 사용하여 재조합 이온 채널의 상대 셀 표면 및 전체 식의 결정

Inherited or de novo mutations in cation-selective channels may lead to sudden cardiac death. Alteration in the plasma membrane trafficking of these ...

Analyzing Cell Surface Adhesion Remodeling in Response to Mechanical Tension Using Magnetic Beads

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both force-sensing molecules at adhesion sites, and force-dependent transcription factors that regulate lineage-specific gene expression and drive phenotypic outputs. However, the signaling networks converting mechanical tension into biochemical pathways have remained elusive. To explore the signaling pathways engaged upon mechanical tension applied to cell surface receptor, superparamagnetic microbeads can be used. Here we present a protocol for using magnetic beads to apply forces to cell surface adhesion proteins. Using this approach, it is possible to investigate not only force-dependent cytoplasmic signaling pathways by various biochemical approaches, but also adhesion remodeling by magnetic isolation of adhesion complexes attached to the ligand-coated beads. This protocol includes the preparation of ligand-coated superparamagnetic beads, and the application of define tensile forces followed by biochemical analyses. Additionally, we provide a representative sample of data demonstrating that tension applied to integrin-based adhesion triggers adhesion remodeling and alters protein tyrosine phosphorylation.

Cell surface adhesions are central in mechanotransduction, as they transmit mechanical tension and initiate the signaling pathways involved in tissue homeostasis and development. Here, we present a protocol for dissecting the biochemical pathways that are activated in response to tension, using ligand-coated magnetic microbeads and force application to adhesion receptors.

자석 구슬을 사용하여 기계 인장에 대한 응답으로 세포 표면 접착 리모델링 분석

Mechanosensitive cell surface adhesion complexes allow cells to sense the mechanical properties of their surroundings. Recent studies have identified both ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

필라멘트 곰팡이의 살아있는 세포 이미징을 위한 막 및 세포벽 선택적 형광 염료의 적용

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...