This work was supported by Inserm. Marion Bouvet is a fellow of the French Ministry for Research and Technology. Annie Turkieh is a fellow granted by European Union FP7 HOMAGE (305507).

이 프로토콜은 우리의 기관 인간 연구 윤리위원회의 지침을 따른다. 건강한 사람의 기증자 버피 코트는 지역 수혈 센터 (릴, 프랑스)에서 얻었다. 버피 코트에서 얻은 샘플은 INSERM 컬렉션 (N ° DC2010-1209)로 선언됩니다. 1. 재료 및 문화 미디어 준비 <ol><li> 1X PBS을 얻었다 멸균 증류수에 10 배의 인산 완충액 식염수 (PBS)에 희석. </li><li> 와 10 % 풀링 된 인간 혈청이없는 겐타 마이신 (40 ㎍ / ㎖) 및 L- 글루타민 (2 mM)을 보충 RPMI 1640 배지를 확인합니다. </li></ol> 단핵구 유래 대 식세포의 2 차 문화 (MDM) <ol><li> 1X PBS와 buffycoat (25 ml)로 희석. 조심스럽게 실온에서 20 분 동안 1,600 XG에 피콜 / leucosep 튜브와 원심 분리기에로드. </li><li> 새로운 튜브로 계면에서 단핵 세포를 수집합니다. 10 ml의 씻을 배는 PBS는 0.1 %의 에틸렌 디아민을 포함 1X1000 XG, 370 XG에 10 분간 각각 160 XG 다음 번 1X PBS 단독 160 XG에 10 분 동안의 연속적인 회 원심 의해 아세트산 (EDTA). </li><li> 혈청 않고 5 ml의 RPMI-1640 배지에서 세포 펠렛을 재현 탁하고 접시 당 1 × 106 세포의 밀도로 35mm 접시에서 세포를 시드. </li><li> 배양기에서 90 분 동안 침전 후 비 부착 세포를 함유하는 상층 액을 제거한다. 1 ml의 PBS로, 단핵구의 3 배를 구성, 부착 세포를 씻으십시오; 그 이전에 혈청 무료 세포에 10 %를 포함하는 새로운 배지 (v / v)의 인간 혈청 1ml를 추가합니다. </li><li> 배양 6 일 후에, 분화 된 대 식세포 대체 (M2)을 수득 재조합 인간 IL-4 (15 ng의 / ㎖)을 첨가 6 일 동안 유지한다. 그리고, M1 식세포를 수득 4 시간 동안 12 일째에 리포 폴리 사카 라이드 (100 NG / ml)로 분화 된 대 식세포를 치료. </li></ol> 2D 전기 영동을위한 M1과 M2 대식 세포 단백질의 3 추출 <ol><li> 세척 macroph나이 25 mM 트리스, 30 mM 트리스 pH를 8, 4 % 3 포함하는 버퍼의 pH 7.4, 스크랩으로 3 회 - [(3- cholamidopropyl) dimethylammonio] -1- 프로판 (CHAPS), 2 M 티오 7 M 요소를. </li><li> -20 ° C에서 얼음과 상점에서 5 분 동안 1.5 ml의 마이크로 원심 튜브에 적합한 혼합기를 사용하여 세포를 Lyse. </li><li> 상업 브래드 포드 시약을 사용하여 단백질 농도를 결정한다. 사용할 때까지 -20 ° C에서 단백질 100 ㎕ 분주을 저장합니다. </li></ol> 4. Isoelectrofocusing 참고 : 어둠 속에서 모든 라벨링 작업을 수행합니다. <ol><li> 37 ° C에서 1 시간 동안 2 mM 트리스 (2- 카복시 에틸) 포스 핀 (TCEP)에 용해 버퍼와 9 μL로 조정 각각의 샘플 (M1 및 M2 대 식세포)의 5 μg의를 줄입니다. </li><li> 0.8 nM의 / μg의 단백질 농도로 M1 및 M2 추출물의 설프 하이 드릴 - 반응성 염료 추출물 시아닌 3 (Cy3가)을 추가한다. M1로 시아닌 5 (Cy5에)를 첨가하고, M2는 0.8 nM의 / μg의 단백질 농도로 추출하여 부화37 ° C에서 30 분. </li><li> 7 M 우레아, 2 M 티오 우레아, 4 % CHAPS, 130 mM의 디티 오 트레이 톨 (DTT) 및 2 % pharmalytes 함유 시료 완충액의 동일한 부피의 첨가로 반응을 중지. </li><li> 믹스 한 손으로 Cy5에 표지 M2 샘플과 M1 샘플을 Cy3에이 표지와 다른 손에 Cy5에 표지 M1 샘플과 M2 샘플을 Cy3에이 표지. </li><li> 고정화의 pH 구배를 재수 (IPG) 스트립 (240mm, pH를 3-10 선형 구배) 7 M 우레아, 등전점에 2 M 티오 4 % CHAPS 초점을 포함하는 버퍼에 표시된 혼합 된 시료 450 μL와 (IEF) 전지 시스템 어떤 전류를 적용하지 않고 24 시간 동안. </li><li> 3 시간, 3 시간 동안 마지막으로 8,000 V 8000 V에 그라데이션에서 6 시간 동안 1,000 V에 그라데이션에서 다음 3 시간 동안 300 볼트 (V)에서 초점을 수행합니다. </li></ol> 5. 두 번째 차원 참고 : 어둠 속에서 모든 전기 작업을 수행합니다. <ol><li> 평형 완충액에서 IPG 스트립 포함 부화10 분 동안 0.1 mM 트리스 - 염산 (pH를 8), 6 M 우레아, 2 % 소듐 도데 실 설페이트 (/ V w) (SDS), 30 % (v / v) 글리세롤을 보내고. </li><li> 12.5 %의 PAGE 젤에 두 번째 차원 (SDS 폴리 아크릴 아마이드 겔 전기 영동 (PAGE))에 대한 평형 IPG 스트립을 전송하고 아가 로스 로우 - 녹는 밀봉. </li><li> 브로 모 페놀 블루 전면 겔의 바닥에 도달 할 때까지 밤새 300 V이어서 70 V의 정전압 Ettan-Daltsix 시스템을 사용하여 20 ° C에서 전기 영동을 수행. </li></ol> 6. 이미지 인식 및 바이오 인포 매틱스 분석 <ol><li> Cy5에 100 ㎛의 화소 크기로 이미지를 산출하기 위해 Cy3에 대한 580분의 532 nm 내지 670분의 633 나노 미터의 여기 / 방출 파장에서 DIGE 이미 저 스캐너 두 낮은​​ 형광 유리판 사이에 캐스팅 스캔 젤. </li><li> 이전에 (12)을 설명하는대로 상용 소프트웨어와 이미지 분석을 수행합니다. </li><li> 계산하고 각 이미지에 자리 볼륨을 정상화. 소품으로 정규화 된 자리 볼륨을 할당각 지점의 총 가치의 ortion 젤에서 검출. </li><li> 두 그룹 (M1 및 M2) 간의 정규화 스폿 볼륨 값을 비교하여 대 식세포의 각 유형에 대한 단백질 스폿 볼륨의 차이를 분석한다. 변화가 1.5 배 (p &lt;0.05, 일방향 ANOVA 분석) 인 경우 상당한 것으로 스폿 볼륨 사이의 차이를 고려한다. </li></ol>

<ol>
	<li>van Ginderachter, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classical+and+alternative+activation+of+mononuclear+phagocytes:+picking+the+best+of+both+worlds+for+tumor+promotion.">Classical and alternative activation of mononuclear phagocytes: picking the best of both worlds for tumor promotion.</a> Immunobiology. 211, (2006).</li><li>Porcheray, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+activation+switching:+an+asset+for+the+resolution+of+inflammation.">Macrophage activation switching: an asset for the resolution of inflammation.</a> Clin. Exp. Immunol. 142, 481-489 (2005).</li><li>Gordon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+activation+of+macrophages.">Alternative activation of macrophages.</a> Nat. Rev. Immunol. 3, 23-35 (2003).</li><li>Mantovani, A., Locati, M., Vecchi, A., Sozzani, S., Allavena, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decoy+receptors:+a+strategy+to+regulate+inflammatory+cytokines+and+chemokines.">Decoy receptors: a strategy to regulate inflammatory cytokines and chemokines.</a> Trends Immunol. 22, 328-336 (2001).</li><li>Pinet, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology,+homogeneity+and+functionality+of+human+monocytes-derived+macrophages.">Morphology, homogeneity and functionality of human monocytes-derived macrophages.</a> Cell. Mol. Biol. 49, 899-905 (2003).</li><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mononuclear+cells+and+granulocytes+from+human+blood.">Isolation of mononuclear cells and granulocytes from human blood.</a> Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1. 97, 77-89 (1968).</li><li>Chinetti-Gbaguidi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+atherosclerotic+plaque+alternative+macrophages+display+low+cholesterol+handling+but+high+phagocytosis+because+of+distinct+activities+of+the+PPAR&#947;+and+LXR&#945;+pathways.">Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPAR&#947; and LXR&#945; pathways.</a> Circ. Res. 108, 985-995 (2011).</li><li>Tonge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+and+development+of+fluorescence+two-dimensional+differential+gel+electrophoresis+proteomics+technology.">Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.</a> Proteomics. 1, 377-396 (2001).</li><li>Shaw, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+saturation+labelling+two-dimensional+difference+gel+electrophoresis+fluorescent+dyes.">Evaluation of saturation labelling two-dimensional difference gel electrophoresis fluorescent dyes.</a> Proteomics. 3, 1181-1195 (2003).</li><li>Dupont, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+saturation+dye+2D+DIGE+proteomics+to+characterize+proteins+modulated+by+oxidized+low+density+lipoprotein+treatment+of+human+macrophages.">Application of saturation dye 2D DIGE proteomics to characterize proteins modulated by oxidized low density lipoprotein treatment of human macrophages.</a> J. Proteome Res. 7, 3572-3582 (2008).</li><li>Dupont, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+maps+and+databases+of+the+human+macrophage+proteome+and+secretome.">Two-dimensional maps and databases of the human macrophage proteome and secretome.</a> Proteomics. 4, 1761-1778 (2004).</li><li>Acosta-Martin, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+incomplete+DNase+I+treatment+on+human+macrophage+analysis.">Impact of incomplete DNase I treatment on human macrophage analysis.</a> Proteomics Clin. Appl. 3, 1236-1246 (2009).</li><li>Acosta-Martin, A. E., Lane, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+bioinformatics+and+MS-based+proteomics:+clinical+implications.">Combining bioinformatics and MS-based proteomics: clinical implications.</a> Expert Rev Proteomics. , (2014).</li><li>Boytard, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+proinflammatory+CD68+MR-+macrophages+in+peroxiredoxin-1+expression+and+in+abdominal+aortic+aneurysms+in+humans.">Role of proinflammatory CD68+MR- macrophages in peroxiredoxin-1 expression and in abdominal aortic aneurysms in humans.</a> Arterioscler. Thromb. Vasc. Biol. 33, 431-438 (2013).</li></ol>

There are no declared conflict of interest.

본원에 기재된 프로토콜은 식세포, M1 (염증 유발)와 M2 (소염)의 두 아형의 다양한 자극의 영향을 분석하는 방법을 자세히. 이전에 7을 발표 한 M1과 M2 대 식세포의 차 문화는 단핵구의 분화에서 얻었다. 2D DIGE 겔 전기 영동 과정은를 Cy3 및 Cy5의 형광 용 스캐너 여기 / 발광 파장 두배 겔을 검사하기 위하여 SDS-PAGE 용 isoelectrofocusing 낮은 형광 플레이트 IEF 셀 같은 특수 재?...

대 식세포는 별개의 기능 표현형을 획득 할 수있는 이기종 및 플라스틱 세포이다. 생체 내,이 세포가 미생물 제품, 사이토 카인 등. 1과 마이크로 환경 signalssuch의 큰 다양성에 응답합니다. 시험관, 염증성 표현형 (M1) 대 식세포의 리포 폴리 사카 라이드 (LPS) 및 인터루킨 -4 (IL-4)와 같은 일부 사이토킨에 의해 항 염증 표현형 (M2)에 의해 유도 될 수있다. 또한, 대 식세포는 특정 신호 2에 따라, 반대로 M2 표현형에 활성화 된 M1에서 전환 할 수 있습니다. 표현형에 따라, 대 식세포는 다른 기능을해야합니다. M1 마크로파지는 미생물 또는 종양 세포 (3)를 죽이고, 종양 괴사 인자 α (TNF-α)와 같은 염증성 사이토 카인을 생산하는 세포이다. 반면, M2 식세포 안티 inflammator를 생성함으로써 상처 치유 및 섬유증과 같은 이러한 염증 반응을 방지3,4-을 TGF-β가 같은 Y 요인. 이전 Boyum 6에서 적응 기술을 사용하여 5 바와 같이 건강한 기증자로부터 인간의 말초 혈 단핵 세포를 피콜 밀도 구배 원심 분리에 의해 분리 하였다. 문화의 대 식세포는 차 문화 (7) 6 일 이후 M1 또는 M2 표현형로 분화 할 수있다. 단백질 발현 또는 숙주 병원성 미생물 또는 독소와 같은 다양한 제어되는 자극 하에서 식세포 두 아형 사이 단백질 변화 분석, 프로 및 소염 식세포의 기능을 해독하는데 도움이 될 것이다. 단백질 체학은 특히 업 또는 다양한 자극에 따라 인간 배양 된 대 식세포에서 아래로 조절되어 단백질의 직접 모니터링을위한 독특한 도구입니다. 형광 염료는 낮은 감도 및 이미지 분석 (8) 등의 2 차원 겔 전기 영동의 한계점을 해결 한 &lt;<html> / SUP&gt;. 시스테인 잔기와 반응 염료는 리신 잔기 (9)와 반응에 비해 검출 감도가 증가하고있다. 이전의 연구에서는 고전 실버 염색 2 차원 전기 영동 (11)에 비해 부족한 시료 (10)의 분석을 위해 DIGE 포화 라벨링의 유용성을 입증 하였다. 이 기술은 빠른 속도로 대 식세포의 두 하위 유형 간 또는 동일한 하위 유형에서 치료 및 치료 대식 세포 사이의 단백질 변형을 분석하는 데 도움이됩니다. 이 단백질체 기술의 장점은 2D 겔 (12)을 분석하여 단백질의 크기 및 번역 후 변형의 정보에 대한 액세스를 가지고있다. 이 분석 될 수있는 샘플의 수를 제한하는, 이것이 높은 처리량 기법 아니다 고려되어야한다. 질량 분석을 기반으로 높은 처리량 분석의 개발 검토로는 최근에 13이을 향상시킬 수 있습니다. _content &quot;&gt; 여기에서는 전기 영동 isoelectrofocusing의 과정을 통해 배양 된 대 식세포의 단백질 추출에서 2D DIGE 분석을 수행하는 방법을 제시하고, 적절한 2D 소프트웨어의 유용성에 대한 SDS-PAGE 정보뿐만 아니라.

<table><tbody><tr><td>RPMI 1640</td><td>Invitrogen</td><td>31870-074</td><td></td></tr><tr><td>PBX 10 X</td><td>Invitrogen</td><td>14200-083</td><td></td></tr><tr><td>L-glutamine-200 mM-100X</td><td>Invitrogen</td><td>25030-024</td><td></td></tr><tr><td>gentamycin 10 mg/ml</td><td>Invitrogen</td><td>15710-049</td><td></td></tr><tr><td>human serum</td><td>Invitrogen</td><td>34005100</td><td></td></tr><tr><td>Ficoll d = 1,077</td><td>ATGC</td><td>L6115</td><td></td></tr><tr><td>Leucosep</td><td>Dutscher</td><td>16760</td><td></td></tr><tr><td>&amp;nbsp;6-wells plate PRIMARIA&amp;nbsp;</td><td>Becton Dickinson</td><td>353846</td><td></td></tr><tr><td>IL-4</td><td>Promocell</td><td>B-61410</td><td></td></tr><tr><td>lipopolysaccharide</td><td>Sigma-Aldrich</td><td>L-2654</td><td></td></tr><tr><td>Giemsa</td><td>Fluka</td><td>48900</td><td></td></tr><tr><td>EDTA MM372,2</td><td>Research Organics&amp;nbsp;</td><td>3.00E+01</td><td></td></tr><tr><td>Filter 0.22 &amp;micro;m</td><td>Millipore</td><td>SCGPTORE</td><td></td></tr><tr><td>100 ml cylinder</td><td>Corning</td><td>430182</td><td></td></tr><tr><td>TCEP</td><td>Interchim</td><td>UP242214</td><td></td></tr><tr><td>Bradford reagent</td><td>Bio-Rad</td><td>5000006</td><td></td></tr><tr><td>Cy3+Cy5-reactive dye</td><td>GE Healthcare</td><td>25-8009-83</td><td></td></tr><tr><td>IPG strip 3-10 24cm</td><td>GE Healthcare</td><td>17-6002-44</td><td></td></tr><tr><td>Protean IEF cell</td><td>Bio-Rad</td><td>165-4000</td><td></td></tr><tr><td>Low-melting agarose</td><td>Invitrogen</td><td>15517-014</td><td></td></tr><tr><td>Ettan-Daltsix system</td><td>GE Healthcare</td><td>80-6485-08</td><td></td></tr><tr><td>Ettan DIGE Imager scanner</td><td>GE Healthcare</td><td></td><td></td></tr><tr><td>Progenesis Samespot</td><td>Non linear dynamics</td><td></td><td></td></tr><tr><td>50 ml tubes</td><td>any supplier</td><td>n/a</td><td></td></tr><tr><td>15 ml tubes</td><td>any supplier</td><td>n/a</td><td></td></tr><tr><td>CHAPS</td><td>Sigma-Aldrich</td><td>C5070</td><td></td></tr><tr><td>Ur&amp;eacute;e</td><td>Merk</td><td>108484-500</td><td></td></tr><tr><td>Thiour&amp;eacute;e</td><td>Sigma-Aldrich</td><td>T7875</td><td></td></tr><tr><td>DTT</td><td>Bio-Rad</td><td>1610611</td><td></td></tr><tr><td>APS</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>T9281</td><td></td></tr><tr><td>Tris Base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Tris HCl</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Pharmalytes 3-10</td><td>GE Healthcare</td><td>17-0456-01</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L3773</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich</td><td>114391</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G6279</td><td></td></tr><tr><td>Acrylamide 40%</td><td>Bio-Rad</td><td>161-0148</td><td></td></tr><tr><td>2D clean Up</td><td>GE Healthcare</td><td>80-6454-51</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td></td></tr><tr><td>Dim&amp;eacute;thylformamide</td><td>Sigma-Aldrich</td><td>22705-6</td><td></td></tr><tr><td>electrode wicks</td><td>Bio-Rad</td><td>165-4071</td><td></td></tr></tbody></table>

proteomic profiling of macrophages by 2d electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

적절한 차동 프로테옴 분석을 수행하기 위해, 분석 할 시료의 처리를 확인해야합니다. 이전 5 공표 제시된 예에서, 대 식세포의 세포 배양 품질은 형태학 및 분자 양태 요구된다. 대 식세포에 단핵구의 분화와 문화의 동질성이 위상 현미경으로 하였다. 그림 1은 M1과 M2 대 식세포의 차 문화의 예를 보여 주었다. 도시 된 바와 같이, 우리는 M1 (그림 1A, ...

Watch this Scientific Journal Video about Proteomic Profiling of Macrophages by 2D Electrophoresis at JoVE.com

Proteomic Profiling of Macrophages by 2D Electrophoresis

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

2D 전기 영동에 의한 대 식세포의 프로테옴 프로파일 링

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

proteomic-profiling-of-macrophages-by-2d-electrophoresis

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

12.6K 조회수.  University Lille Nord de France. Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

비디오: Proteomic Profiling of Macrophages by 2D Electrophoresis

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.

Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.

2 차원 젤 전기 이동 법 질량 분석 방법으로 인간의 뇌 Adenoma 조직 프로테옴의 분석에 대 한 결합

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

연구

암 연구학

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

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 almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1). Protein-protein interactions are intrinsic to signaling pathways, and to gain more insight in these complex biological processes, sensitive and reliable methods are needed for studying cell surface proteins. Two-dimensional (2-D) electrophoresis is used extensively for detection of biomarkers and other targets in complex protein samples to study differential changes. Cell surface proteins, partly due to their low abundance (1 2% of cellular proteins), are difficult to detect in a 2-D gel without fractionation or some other type of enrichment. They are also often poorly represented in 2-D gels due to their hydrophobic nature and high molecular weight (2). In this study, we present a new protocol for intact cells using CyDye  DIGE Fluor minimal dyes for specific labeling and detection of this important group of proteins. The results showed specific labeling of a large number of cell surface proteins with minimal labeling of intracellular proteins. This protocol is rapid, simple to use, and all three CyDye DIGE Fluor minimal dyes (Cy 2, Cy 3 and Cy 5) can be used to label cell-surface proteins. These features allow for multiplexing using the 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE) with Ettan  DIGE technology and analysis of protein expression changes using DeCyder  2-D Differential Analysis Software. The level of cell-surface proteins was followed during serum starvation of CHO cells for various lengths of time (see Table 1). Small changes in abundance were detected with high accuracy, and results are supported by defined statistical methods.

A simple and specific method was demonstrated for fluorescent labeling and enhanced detection of cell surface proteins without a fractionation step. Differential abundance in cell surface proteins was analyzed using two-dimensional (2-D) electrophoresis and Ettan&#x2122; DIGE technology.

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 ...

생물학

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

낮은 산소 환경에서 인간 대 식 세포 분극의 Proteomic 분석

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

생화학

Whole-cell MALDI-TOF Mass Spectrometry is an Accurate and Rapid Method to Analyze Different Modes of Macrophage Activation

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and biomarkers. Recently, it has been used in microbiology for the routine identification of bacteria grown from clinical samples, without preparation or fractionation steps. We and others have applied this whole-cell MALDI-TOF mass spectrometry technique successfully to eukaryotic cells. Current applications range from cell type identification to quality control assessment of cell culture and diagnostic applications. Here, we describe its use to explore the various polarization phenotypes of macrophages in response to cytokines or heat-killed bacteria. It allowed the identification of macrophage-specific fingerprints that are representative of the diversity of proteomic responses of macrophages. This application illustrates the accuracy and simplicity of the method. The protocol we described here may be useful for studying the immune host response in pathological conditions or may be extended to wider diagnostic applications.

This protocol describes the use of whole-cell MALDI-TOF mass spectrometry on eukaryotic cells. Here, we illustrate the accuracy of this technique by analyzing the multiple activation states of macrophages in response to their microenvironment.

전체 셀 MALDI-TOF 질량 분석은 대 식세포의 활성화의 다양한 모드를 분석 할 수있는 정확하고 빠른 방법입니다

MALDI-TOF is an extensively used mass spectrometry technique in chemistry and biochemistry. It has been also applied in medicine to identify molecules and ...

면역학 및 감염병학

Consensus Brain-derived Protein, Extraction Protocol for the Study of Human and Murine Brain Proteome Using Both 2D-DIGE and Mini 2DE Immunoblotting

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or pathological conditions. Basically, this technique is based on the separation of proteins according to their isoelectric point in a first step, and secondly according to their molecular weights by SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this report an optimized sample preparation protocol for little amount of human post-mortem and mouse brain tissue is described. This method enables to perform both two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mini 2DE immunoblotting. The combination of these approaches allows one to not only find new proteins and/or protein modifications in their expression thanks to its compatibility with mass spectrometry detection, but also a new insight into markers validation. Thus, mini-2DE coupled to western blotting permits to identify and validate post-translational modifications, proteins catabolism and provides a qualitative comparison among different conditions and/or treatments. Herein, we provide a method to study components of protein aggregates found in AD and Lewy body dementia such as the amyloid-beta peptide and the alpha-synuclein. Our method can thus be adapted for the analysis of the proteome and insoluble proteins extract from human brain tissue and mice models too. In parallel, it may provide useful information for the study of molecular and cellular pathways involved in neurodegenerative diseases as well as potential novel biomarkers and therapeutic targets.

A common protein extraction protocol using urea/thiourea/SDS buffer for human and mice brain tissue allows indentification of proteins by 2D-DIGE and their subsequent characterization by mini 2DE immunoblotting. This method enables one to obtain more reproducible and reliable results from human biopsies and experimental models.

합의 뇌 유래 단백질, 2D-DIGE 미니 2DE 면역 블롯을 모두 사용하여 인간과 쥐과 뇌 프로테옴 연구를 위해 추출 프로토콜

Two-dimensional gel electrophoresis (2DE) is a powerful tool to uncover proteome modifications potentially related to different physiological or ...

신경과학

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as &#8220;spots&#8221; on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.

Here we describe a protocol that couples two proteomic techniques, namely 2-dimensional Electrophoresis (2DE) and Mass Spectrometry (MS), to identify differentially expressed/post-translational modified proteins among two or more groups of primary samples. This approach, together with functional experiments, allows the identification and characterization of prognostic markers/therapeutic targets.

단백질 기능에 2DE - 젤 지점에서 만성 림프 구성 백혈병에 HS1에서 배운 레슨

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease&#8217;s biology and, as a ...

의학

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

셀룰러 Lysates에서 Multiprotein의 단지의 분석을 위해 블루 기본 Polyacrylamide 젤 전기 영동 (BN - PAGE)

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

절연, 특성화 및 비만 관련 염증에 의해 영향을 받는 조직에서 세포의 정화

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Label-Free Quantitative Proteomics Workflow for Discovery-Driven Host-Pathogen Interactions

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s global proteome under varying conditions. This powerful strategy applied to the interactions of microbial pathogens with the desired host comprehensively characterizes both perspectives towards infection. Herein, the workflow describes label-free quantification (LFQ) of the infectome of Cryptococcus neoformans, a fungal facultative intracellular pathogen that is the causative agent of the deadly disease cryptococcosis, in the presence of immortalized macrophage cells. The protocol details the proper protein preparation techniques for both pathogen and mammalian cells within a single experiment, resulting in appropriate peptide submission for liquid-chromatography (LC)-MS/MS analysis. The high throughput generic nature of LFQ allows a wide dynamic range of protein identification and quantification, as well as transferability to any host-pathogen infection setting, maintaining extreme sensitivity. The method is optimized to catalogue extensive, unbiased protein abundance profiles of a pathogen within infection-mimicking conditions. Specifically, the method demonstrated here provides essential information on C. neoformans pathogenesis, such as protein production necessary for virulence and identifies critical host proteins responding to microbial invasion.

Here, we present a protocol to profile the interplay between host and pathogen during infection by mass spectrometry-based proteomics. This protocol uses label-free quantification to measure changes in protein abundance of both host (e.g., macrophages) and pathogen (e.g., Cryptococcus neoformans) in a single experiment.

발견 중심의 숙주-병원균 상호 작용을 위한 라벨 프리 정량적 프로테오믹스 워크플로우

The technological achievements of mass spectrometry (MS)-based quantitative proteomics opens many undiscovered avenues for analyzing an organism&#8217;s ...

Two-Dimensional Gel Electrophoresis

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct separation methods that have been coupled together: isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This physically separates compounds across two axes of a gel by their isoelectric points (an electrochemical property) and their molecular weights.
The procedure in this video covers the main concepts of 2DGE and a general procedure for characterizing the composition of a complex protein solution. Three examples of this technique are shown in the applications section, including biomarker detection for disease initiation and progress, monitoring treatment in patients, and the study of proteins following posttranslational modification (PTM).
<div class="chapter_text" id="script_0">
Two-dimensional, or 2D, gel electrophoresis is a technique utilizing two distinct separation methods which can separate thousands of proteins from a single mixture. One of the techniques, SDS-PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis, cannot fully separate complex mixtures alone. 2D gel electrophoresis couples the SDS-PAGE to a second method, isoelectric focusing or IEF, which separates based on isoelectric points, allowing for the resolution of potentially all proteins in a cell lysate. This video will show the principles of 2D gel electrophoresis, a general procedure, and some of its biomedical applications.
</div>
<div class="chapter_text" id="script_1">
2D gel electrophoresis begins with IEF as the first dimension. Every protein has a pH value, called the isoelectric point or pI, where the net charge is zero. When a protein is subjected to an electric field, it will move toward the electrode with opposite charge. Samples of interest are loaded onto immobilized pH gradient, or IPG, strips which have embedded ampholytes, molecules containing both acidic and basic groups. An electric field is then applied to the pH gradient strip, causing the proteins to migrate until they reach the pH value matching their pI, where they lose their net charge.
Prior to running the second-dimension, the embedded proteins are treated with SDS, denaturing them and providing a uniform negative charge. Once completed, the IPG strips are placed onto a polyacrylamide gel. An applied electric field draws the proteins toward the anode, with larger proteins moving more slowly through the gel.
Once the protein mixture has been separated according to pI and molecular weight, the proteome map is visualized using stains, and proteins of interest are identified.
Now that we have discussed the principles of 2D gel electrophoresis, let&#39;s go over a typical laboratory procedure.
</div>
<div class="chapter_text" id="script_2">
Before the experiment can be performed, the proteins must be solubilized into media. Solubilization of the sample is achieved by de-aggregating proteins with a combination of chaotropic agents for disrupting hydrogen-bonding interactions, nonionic detergents to prevent altering of the proteins&#39; charge, reducing agents to break disulfide bonds, and buffers. To remove interfering abundant proteins and other molecules, the material is sequentially extracted by centrifugation, and collection of the resulting pellet; followed by treatment with endonuclease, an enzyme used to consume any DNA that would interfere with the experiment.
Once the proteins have been solubilized, IPG strips are prepared by rinsing with a strip-cleaning solution and left upside-down to dry. Each strip is then assigned a strip holder number. Once ready, the cellular extract is loaded onto the strips in a slow, sliding motion from the negative to the positive end. For the purpose of rehydration, damp blotting paper is placed on top of the electrode and under the gel strips; the IPG strips are then lined onto the IEF instrument. A high electric current is applied, and the proteins begin to migrate.
</div>
<div class="chapter_text" id="script_3">
Following completion of the first dimension, the gel for SDS-PAGE is prepared in a casting apparatus. The IPG strips are treated by placing them face down in SDS-containing equilibration buffer. The electrophoresis unit is readied with the addition of electrophoresis buffer. The treated IPG strips are collected using tweezers, placed on top of the gel plates, and sealed with agarose-sealing solution. A voltage source then applies an electric field, which is held until the fastest-moving proteins are 1 cm from the bottom of the gel.
After completion of the electrophoresis, the proteins must be visualized. Traditionally this is performed by staining with Coomassie blue or silver nitrate. Proteins of interest may be transferred from the gel, and analyzed by Western blot analysis.
A second identification approach involves excision of the proteins from the gel, digesting them, than analyzing them by mass spectrometry.
</div>
<div class="chapter_text" id="script_4">
Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the uses for 2D gel electrophoresis.
One of the most common uses for this technique is the identification of molecules involved in disease initiation and progression. 2D gel electrophoresis, coupled with mass spectrometry, can detect the up- or down-regulation of specific proteins in diseased areas in comparison to healthy ones.
Additionally, 2D gel electrophoresis is useful in following the progress of patients&#39; response to a potential therapeutic drug. Specimens may be taken from patients at various timepoints following administration of the treatment. In this way, 2D gel electrophoresis coupled with Western blot or mass spectrometry analysis, can detect proteins associated with negative responses such as inflammation; or the absence of proteins in an alleviated state.
Another use for 2D gel electrophoresis is in the study of protein structure and function following posttranslational modification, or PTM, which are additions to proteins following their translation from mRNA. PTM&#39;s can regulate a variety of functions, including protein signaling, gene expression, or cause oxidative damage. 2D gel electrophoresis is sensitive to modifications such as methylation or acetylation, which can cause a shift in pI as well as the molecular weight.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on 2D gel electrophoresis. This video described the principles of the technique, a typical experimental procedure, and several of its applications in the field of biomedicine.
Thanks for watching!
</div>

2차원 젤 전기포레시스

Two-dimensional gel electrophoresis (2DGE) is a technique that can resolve thousands of biomolecules from a mixture. This technique involves two distinct ...