저자는 크게 피에르 Bourdoncle, 토마스 길버트와 코친 이미징 시설의 루이스 Rimbault을 인정합니다. 이 작품은 INSERM, CNRS와 리그 국립 contre 르 암 (퀴프의 labellisée)에 의해 지원되었다.

T 림프구의 1. 준비 <ol><li> 10, 11 설명 된대로 기본 마우스 또는 인간 T 세포를 준비합니다. </li><li> 3 × 106 세포 / ml - (2)의 농도에서 10 % 인간 AB 혈청 완전한 RPMI 배지에서 인간 T 세포를 배양. 주 : 배양 인간 T 세포의 많은 부분의 자발 분극을 유도 할 수 소 태아 혈청 (FCS) 대신에 인간 혈청을 사용. 이 기간 동안 언제든지 5 일 추가로 과정을 - 4 문화에서 이러한 세포를 보관하십시오. </li><li> 유사한 세포 밀도로 10 % FCS가 보충 된 완전 RPMI 배지에서 마우스 T 세포를 유지한다. 10 NG / ㎖ IL7의 존재하에 37 ℃에서 즉시 또는 다음 날, O / N에서 배양 한 후 사용한다. </li><li> 완전한 RPMI 10 % FCS로 보충 된 CEM 인간 T 세포주를 배양. </li></ol> 2. 케모카인 자극 <ol><li> 10 mM의 Hepe 보충 따뜻한 HBSS 매체에 실험 조건 당 5 × 10 5 세포를 씻으의. 몇 가지 실험을 위해, 2 μg의 pmaxGFP 8 μg의 한 pcDNA3.1 + (빈 벡터 제어), 죽은 PKCζ 키나아제, 또는 N- 말단 Par6 플라스미드로 전날 차 인간 T 세포를 공동 형질. </li><li> 따뜻한 HBSS - 헤 페스 배지에서 세포 펠렛을 재현 탁 (실험 조건 0.3 ㎖의 X 번호를 사용). </li><li> 1.5 ml의 마이크로 원심 튜브에 세포를 배포합니다. 주 : 따라서 단일 실험 조건에 대응 한 튜브를 0.3 ㎖의 HBSS - 헤 페스 매체에 5 × 105 세포를 포함 할 것이다. 일부 실험에서, 500 nM의 Latrunculin 또는 비히클 (DMSO)를 첨가하고, 케모카인 자극 전에 30 분 동안 세포를 배양한다. </li><li> 각 튜브에 10 NG / ㎖의 CCL19 또는 CXCL12 케모카인 (500)에 추가합니다. 참고 : 우리는 보통 10 사용 - 인간 T 세포에 대한 200 NG / ml의 케모카인을. CEM 세포는 CCL19 결합 CCR7 수용체를 발현하지 않습니다. 따라서, CEM 세포 만 CXCL12 자극에 응답 할 수있다. 500 N - 또한, 높은 케모카인 농도 ((300)를 사용하여g / ml)를 마우스 T 세포를 편광 시키. </li><li> 튜브를 수회 뒤집고 각각 일차 T 세포 또는 CEM을 8 또는 10 분 동안 37 ° C까지 수조에서이를 부화. </li><li> 각 튜브에 2 % 파라 포름 알데히드 (PFA)와 PBS의 10 mM의 Hepes를 함유 따뜻한 용액 0.3 ㎖를 첨가하여 분극 처리를 중지. 튜브를 뒤집어 5 분 동안 37 ° C에서 그들을 부화. </li></ol> 3. 염색과 <ol><li> 튜브 유동 세포 계측법하기 위해 마이크로 원심 튜브에서 세포를 전송합니다. </li><li> 1 % 소 혈청 알부민 (BSA), 0.5 mM의 EDTA PBS 및 10 mM의 Hepes를 함유 RT 용액으로 완전히 관을 채운다. </li><li> 4 분 동안 460 XG에 튜브를 원심 분리기. </li><li> 상층 액을 제거하고 PBS에서 5 % FCS를 함유 RT 용액 100 ㎕에 세포를 재현 탁. </li><li> 멀리 빛, 소용돌이를, 각 튜브에 FITC-항 HLA-ABC의 5 μl를 추가하고, 실온에서 30 분 동안 그들을 품어. </li><li> , PBS 5 %를 FCS를 함유 한 번 세포를 씻으한 번만 PBS와 ND. </li><li> RT에서 : 다음 1 % BSA, 0.5 mM의 EDTA와 PBS에서 10 mM의 HEPES를 포함하는 용액으로 세포를 씻어 10 분 동안 품어, 500 ㎕의 1 % PFA를 추가합니다. </li><li> 상층 액을 버리고 PBS는 0.2 % BSA, 0.1 % 사포닌, 0.5 mM의 EDTA 10 mM의 헤 페스 (투과성으로 버퍼)를 포함하는 용액으로 완전히 튜브를 입력합니다. </li><li> 이 매체에 두 번 세포를 씻으십시오. </li><li> 배지를 제거하고 세포 펠릿을 해리하도록 와류 튜브 플립. </li><li> 각각의 튜브에 방지 P-ERM 항체의 100 희석, 소용돌이를하고 실온에서 30 분 동안 품어 : 및 / 또는 1 0.25 ㎍ / ㎖의 TRITC-Phalloidin의를 추가합니다. </li><li> 투과성으로 버퍼와 세포를 씻으십시오. 형광 이차 항체로 인큐베이션 필요한 경우. </li><li> 200 μl의 PBS에 세포를 재현 탁하고 마이크로 원심 튜브로 전송. 참고 : 세포는 획득을위한 준비가되어 있습니다. 튜브에 200 μL의 최대 총 부피를 유지하면서 선택적으로, 최종 1 %로 농축시켜 추가 PFA세포는 추가 처리를 위해 당일 사용되지 않아야하는 경우, 순서의 염색 농도를 유지한다. </li></ol> 4. 이미지 수집 및 분석 <ol><li> 장치 (특정 시약 / 장비의 표 참조)로 전환하고 제조업체의 지침에 따라 자체를 교정 할 수 있습니다. 참고 : INSPIRE 소프트웨어는 40 배 목표 (0.75 NA)로 사용되었다. IDEAS 3.0 소프트웨어는 모든보고 정량화에 사용되었다. </li><li> 미래의 실험 템플릿에 저장할 수 있습니다 획득 일련의 창을 준비합니다. RMS 그라데이션 값을 정량화 히스토그램을 그립니다. RMS 그라데이션 막대 그래프에서 선택한 &quot;초점에서&quot;인구에서, 지역의 히스토그램에서 &quot;단일 셀&quot;인구 경계를 정하다. 마이크로 원심 튜브가 기기에 삽입되면, 각 셀에 대한 게이트 레이저 파워를 조정하고 5000 T 림프구를 취득. </li><li> 부드러운 아이디어도자기, 인수 파일을 열고 각 셀에 대해 얻은 밝은 필드 이미지의 그라데이션 RMS 값의 값을 표시하는 막대 그래프 (채널 1)을 만듭니다. 57 위의 RMS에게 그라데이션 값을 나타내는 세포를 고려 RMS 그라데이션 히스토그램에 게이트를 그립니다. 참고 : RMS 구배 분석 초점면에있는 경우에만 T 림프구에서 고려 허용합니다. 우리는 (57)에 우수한 그라데이션 RMS 값을 나타내는 각각의 세포는 초점면에 있고, 정확하게 고려 될 수 있음을 경험적으로 결정했다. </li><li> 분석 / 마스크 메뉴에서, 2 픽셀의 침식 시야 이미지 M01,의 형태 마스크에서 (M01,2)을 침식라는 새로운 마스크를 만들 수 있습니다. 그런 다음, 분석 / 침식 (M01,2) 마스크 계산 영역의 새로운 기능을 만들 메뉴를 갖추고 있습니다. 게이트 이전에 선택된 셀로부터,이 새로 만든 마스크를 이용하여 상기 셀 영역을 나타내는 히스토그램을 연다. 참고 :에 의해 사용할 수있는 형태의 마스크디폴트는 셀의 실제 크기보다 훨씬 더 크다. 따라서,이 화소로까지 침식 더 정확하다. </li><li> 교정 구슬과 이물질 (작은 지역)과 이중선 (대형 지역)을 제외한 개별 T 세포에 게이트를 그립니다. 각 인수에서이 게이트를 조정합니다. 주 : 셀 크기에 변동이 게이트의 위치에 영향을 미칠 것이다. 마우스 T 세포가 스스로 CEM 세포보다 작은 인간 T 세포보다 작은 같이, 각 세포 유형의 면적의 크기에 비례한다. </li><li> 이전 단계에서 게이트 된 하나에 초점 세포를 감안하면, 팔로이 딘의 원시 최대 픽셀의 함수로 HLA-ABC 염색에 원시 최대 픽셀의 (채널 2, X 축)로 구성되는 점 음모를 엽니 다 염색 (채널 4, Y 축). 흠 또는 포화 형광 이벤트를 제외 게이트를 만듭니다. 원시 최대 HLA-ABC를위한 픽셀 (채널 2) 팔로이 딘 (채널 4)의 염색을 보여주는 열기 두 히스토그램. </li><li> 분석 / 마스크 메뉴, CREAT에서2 픽셀의 침식 HLA-ABC 염색 된 세포 (채널 2)의 형태 마스크에서 (M02,2)을 침식라는 개 새로운 마스크. 그런 다음, 분석 / 침식 (M02,2)에 계산 된 원형 매개 변수로 구성된 새로운 기능을 생성, 메뉴 특징. 참고 :이 매개 변수는 원에서 셀 모양의 편차를 측정합니다. 따라서, 완전한 원형 T 세포는 낮은 원형도 지수를 나타낼 것이다 편광 신장 세포 반면 높은 원형도 값을 가질 것이다. </li><li> 이어서, 이전 셀로부터 게이트 된 원형 형상의 값을보고 다른 히스토그램을 생성한다. 직접 각각의 개별 셀에 대한 원형도 값에 따라 개인 합초 세포군의 분포를 플롯이 히스토그램을 사용한다. </li><li> 비 - 자극 된 세포에 대한 히스토그램의 형상을 살펴보면, 원형 제한값까지 낮은 원형도 값에서 시작하여, 편광 된 셀을 그리 게이트 여기서 비 자극 된 세포의 대부분플롯됩니다. 게이트 인구 중 편광 세포의 비율에 대한 통계의 디스플레이를 확인합니다 (%는 정문). 참고 : 우리는 13 아래 원형 인덱스 값이 게이트를 설정했습니다. </li><li> 세포에서 액틴 염색 편광보고하기 위하여, HLA-ABC 염색 (채널 2) 및 팔로이 딘 염색을 비교 밝은 세부 비슷한 R3 값에 대한 히스토그램을 플롯 (채널 4). 주 :이 기능의 더 큰 값이 더 적 착색 개의 중첩이다. </li><li> 이전과 같은 게이팅 전략을 사용하여, 편광 액틴 염색 세포의 백분율을 나타낸다 분리 염색 비 자극 된 세포에 게이트 플롯. </li><li> 완료되면, MHC 클래스 I 및 팔로이 딘 염색에 함께 SD ± 모든 세포의 원형과 비슷한 인덱스의 평균값으로 편광 된 세포의 비율의 분포에 편석을 나타내는 세포의 비율은 corresp에 나타낸다onding 통계 패널. </li></ol>

<ol>
	<li>Rossi, D., Zlotnik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+chemokines+and+their+receptors.">The biology of chemokines and their receptors.</a> Annu Rev Immunol. 18, 217-242 (2000).</li><li>Thelen, M., Stein, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+chemokines+invite+leukocytes+to+dance.">How chemokines invite leukocytes to dance.</a> Nat Immunol. 9 (9), 953-959 (2008).</li><li>Rougerie, P., Rho Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases:+masters+of+T+lymphocyte+migration+and+activation.">GTPases: masters of T lymphocyte migration and activation.</a> Immunol Lett. 142 (1-2), 1-13 (2012).</li><li>Sanchez-Madrid, F., del Pozo, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+polarization+in+cell+migration+and+immune+interactions.">Leukocyte polarization in cell migration and immune interactions.</a> Embo J. 18 (3), 501-511 (1999).</li><li>Adams, D. H., 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=Hepatocyte+growth+factor+and+macrophage+inflammatory+protein+1+beta:+structurally+distinct+cytokines+that+induce+rapid+cytoskeletal+changes+and+subset-preferential+migration+in+T+cells.">Hepatocyte growth factor and macrophage inflammatory protein 1 beta: structurally distinct cytokines that induce rapid cytoskeletal changes and subset-preferential migration in T cells.</a> Proc Natl Acad Sci U S A. 91 (15), 7144-7148 (1994).</li><li>Rougerie, P., 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=Fam65b+is+a+new+transcriptional+target+of+FOXO1+that+regulates+RhoA+signaling+for+T+lymphocyte+migration.">Fam65b is a new transcriptional target of FOXO1 that regulates RhoA signaling for T lymphocyte migration.</a> J Immunol. 190 (2), 748-7455 (2013).</li><li>Real, E., Faure, S., Donnadieu, E., Delon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+Atypical+PKCs+regulate+T+lymphocyte+polarity+and+scanning+behavior.">Cutting edge: Atypical PKCs regulate T lymphocyte polarity and scanning behavior.</a> J Immunol. 179 (9), 5649-5652 (2007).</li><li>Negulescu, P. A., Krasieva, T. B., Khan, A., Kerschbaum, H. H., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+of+T+cell+shape,+motility,+and+sensitivity+to+antigen.">Polarity of T cell shape, motility, and sensitivity to antigen.</a> Immunity. 4 (5), 421-430 (1996).</li><li>Zuba-Surma, E. K., Kucia, M., Abdel-Latif, A., Lillard, J. W., Ratajczak, M. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ImageStream+System:+a+key+step+to+a+new+era+in+imaging.">The ImageStream System: a key step to a new era in imaging.</a> Folia Histochem Cytobiol. 45 (4), 279-290 (2007).</li><li>Matheu, M. P., Cahalan, M. D. <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+CD4++T+cells+from+mouse+lymph+nodes+using+Miltenyi+MACS+purification.">Isolation of CD4+ T cells from mouse lymph nodes using Miltenyi MACS purification.</a> J Vis Exp. (9), 409 (2007).</li><li>James, E. A., LaFond, R., Durinovic-Bello, I., Kwok, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+antigen+specific+CD4++T+cells+using+MHC+class+II+tetramers.">Visualizing antigen specific CD4+ T cells using MHC class II tetramers.</a> J Vis Exp. (25), (2009).</li><li>Wabnitz, G. H., 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=L-plastin+phosphorylation:+a+novel+target+for+the+immunosuppressive+drug+dexamethasone+in+primary+human+T+cells.">L-plastin phosphorylation: a novel target for the immunosuppressive drug dexamethasone in primary human T cells.</a> Eur J Immunol. 41 (11), 3157-3169 (2011).</li><li>Freeley, M., 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=L-plastin+regulates+polarization+and+migration+in+chemokine-stimulated+human+T+lymphocytes.">L-plastin regulates polarization and migration in chemokine-stimulated human T lymphocytes.</a> J Immunol. 188 (12), 6357-6370 (2012).</li></ol>

저자는 그들이 더 경쟁 금융 이익이 없다는 것을 선언합니다.

계측법, 신속하고 유익한 게이팅 전략 케모카인 자극에 의​​해 유도 된 세포 및 분자 이벤트를 분석하는 촬상 흐름의 최신 기술을 사용하는 것이 제시된다. 케모카인 자극 분극 공정 동안 서로 다른 단백질의 세포 내 분포에 의해 유도 된 세포 형태의 변화 : 하나의 실험에서, 하나는 정보의 두 가지 유형을 얻을 수있다. 흥미로운 증거가 통계적 견고성과 전체 세포 집단의 작은 부분의 분석이 ?...

케모카인 전문 위치 1에 세포를 유치 작은 수용성 단백질이다. 따라서, 그들은 조직에서 세포의 정확한 위치, 개발 및 생리에 중요한 기능에 참여하고 있습니다. 이 효과적인 면역 반응을 탑재 콘서트 행동 많은 다른 종류의 세포의 작용에 의존으로 면역 시스템은이 규칙에 예외는 아닙니다. 외부 항원이 검출 중화되기 전에 주어진 상태에서 하나의 면역 세포 유형의 특정 위치를 제어함으로써, 케모카인 장치들이 필요하다. 특히 T 림프구에서, 케모카인은 T 세포 운동성 2,3- 호의 결합시, 다양한 세포 내 신호를 유도하는, 특정한 표면 수용체 (칼슘 증가, ERK 인산화의 Rho GTP 아제 활성, 인테그린 친화 세포 골격 변화의 증가)에 결합한다. 세포 수준에서, 하나는 케모카인 유도 자극에 형태 학적 변화를 관찰 할 수있다.세포 모양에서 이러한 변화는 T 세포에서 특히 극적 : 혈류에 여행 할 때 휴식 T 세포가 구슬처럼 둥근 형태를 가지고있다. 앞에 리딩 에지​​ : 그러나, 염증 부위에 또는 림프 기관의 근방에서 케모카인의 존재를 감지 해주기 양극성 형태로 이루어진 전형적인 &quot;핸드 미러&quot;형태를 채택하는 T 세포의 모양을 변경하려고 그리고 다시 4에서 트레일 링 에지, 또는 uropod. 또한, 균체 내 성분은 마이그레이션을 유지 편광 T 세포의 두 대향하는 영역으로 분리 할 수​​있다. 예를 들면, 케모카인 자극 5 및 액틴 중합시 액틴 필라멘트 중합 증가 편광 된 T 세포 (2)의 전방에 축적한다. 한편, 이러한 피질 F 액틴 세포 골격에 세포막 링크 Ezrin-Radixin-Moesin (ERM)과 가족의 인산화 된 단백질과 같은 여러 가지 단백질의 편광 uropod에 다시 문 지역화T 세포 (6). 흥미롭게도, 우리 등이 분극 처리는 T 세포의 이주를 위해 필요하다는 것을 보여 주었다. 실제로, 편광을 방해하는 어떤 치료는 세포의 운동성을 억제합니다. 예를 들어, 비정형 단백질 구성원의 활성의 억제는 C (PKC) 계열, 및 PKCζ PKCι 블록 T 세포와 수지상 세포 편광 7들의 이동성 스캔 과정 키나아제. T 세포 분극도의 Rho GTP 아제에 의해 조절된다. 우리는 최근에 기재된 Fam65b에서 RhoA 의한 단백질 활성의 조절이없이 Transwell 분석 6 마이그레이션 T 세포 형태의 변화 및 능력을 방해 것을 보여 주었다. 편광 세포 운동성위한 필수 단계로서, 케모카인은 응답의 메인 리드로 정량화 할 수있는 것이 결정적으로 중요하다. 세포 모양의 변화는 이전에 수동으로 8 측정 하였다. 그러나, 이러한 종류의 정량은 매우 시간 소모적이며, 그 보통 몇 그래서세포의 수만 고려된다. 여기서, 새로운 방법이 급속 케모카인 자극에 노출 된 T 림프구의 형상 변화의 정도를 정량화하기 위해 제시된다. 기술 계측법 촬상 유동은 케 모킨 자극 상이한 조건에서 효율적으로 편광 된 세포의 양을 정량화하는 유세포 및 현미경 (9)의 이점을 결합하는 데 사용된다 (특정 시약 / 장비의 도표 참조). 하나의 기술이 견고하게 측정 할 수있는 형태 학적 변화의 정량화에 또한, 케모카인 시그널링시 일부 단백질의 세포 내 위치 변화를 평가하는 것도 가능하다.

<table border="0" cellpadding="0" cellspacing="0" width="569">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:140px;">Name of the material/Equipment</td>
			<td style="width:95px;">Company</td>
			<td style="width:80px;">Catalog number</td>
			<td style="width:255px;">Comments/Description (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">RPMI</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">61870-010</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:140px;">Human serum AB</td>
			<td style="width:95px;">PAA</td>
			<td style="width:80px;">C11-021</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:140px;">Fetal calf serum</td>
			<td style="width:95px;">PAN Biotech</td>
			<td style="width:80px;">P30-3300</td>
			<td style="width:255px;">Pre-heat to inactivate the complement.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:140px;">Human T cell nucleofactor kit</td>
			<td style="width:95px;">Lonza</td>
			<td style="width:80px;">VCA-1002</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Murine IL7</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">217-17</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:140px;">HBSS</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">14025-050</td>
			<td style="width:255px;">Warm in 37 &#176;C water bath before use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Hepes</td>
			<td style="width:95px;">Gibco</td>
			<td style="width:80px;">15630-056</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:140px;">Murine CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">250-27B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:140px;">Human CCL19</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-29B</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells.</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:140px;">Human CXCL12</td>
			<td style="width:95px;">Peprotech</td>
			<td style="width:80px;">300-28A</td>
			<td style="width:255px;">Aliquots are thawed on ice before adding the chemokine to the cells. This chemokine can also be used on mouse T cells.</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:140px;">PFA</td>
			<td style="width:95px;">Electron Microscopy Sciences</td>
			<td style="width:80px;">157-8-100</td>
			<td style="width:255px;">This is a 8 % PFA solution in water. Mix volume to volume with 2X PBS to obtain a 4 % PFA solution in PBS.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">&#160;BSA</td>
			<td style="width:95px;">Sigma</td>
			<td style="width:80px;">A3059</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:140px;">Saponin</td>
			<td style="width:95px;">Fluka</td>
			<td style="width:80px;">84510</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Alexa Fluor 594 phalloidin</td>
			<td style="width:95px;">Invitrogen</td>
			<td style="width:80px;">A12381</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">FITC-anti-HLA-ABC antibody</td>
			<td style="width:95px;">Beckman Coulter</td>
			<td style="width:80px;">IM1838</td>
			<td style="width:255px;">clone B9.12.1</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:140px;">Anti-P-ERM antibody</td>
			<td style="width:95px;">Cell Signaling Technology</td>
			<td style="width:80px;">3149P</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:140px;">ImageStreamx Mark II</td>
			<td style="width:95px;">Amnis</td>
			<td style="width:80px;"></td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

rapid and robust analysis of cellular and molecular polarization induced by chemokine signaling

세포는 과정이라고 편광 자신의 원형을 잃고으로 많은 단백질의 세포 내 현지화를 변경하여 자극을 케모카인에 응답합니다. 고전 이미징 기술은 이러한 현상을 연구하는데 사용되어왔다. 그러나 이러한 형태 및 세포의 수만 염색 공동 지역화 시간 소모적 정량화 하였다 많은​​ 세포의 수동 요구를 취득. 여기서, 신속하고 강력한 방법은 사이토의 것과 현미경의 장점을 결합한 기술 계측법 촬상 흐름을 이용하여 상기 셀 수천 이루어진 샘플들에 이러한 현상을 연구하기 위해 설명된다. MHC 클래스 I 분자와 사상 액틴 용 CCL19 염색 자극 T 림프구를 사용하여, 게이팅 전략 동시에 형상 변화의 정도 및 CCL19 시그널링에 의해 영향 마커의 공동 국산화 정도를 측정하기 위해 제공된다. 또한,이 게이팅 전략은 observ 우리를 허용전자 CXCL12 자극 후 편광 T 세포 (전면) 사상 액틴과 (후면) 인산화 Ezrin-Radixin-Moesin (포스 ERM) 단백질의 분리. Par6 / 비정형 PKC 신호 전달 경로의 돌연변이 발현 억제제 및 약리학 적으로 액틴 중합 억제제 등이 기술은 두 개의 서로 다른 편광 소자에 차단 효과를 관찰하는데 유용 하였다. 따라서, 증거는이 기술은 단백질의 형태 학적 변화 및 ​​재분배 모두를 분석하는 유용한 것을 알 수있다.

여기에서 선택된 첫 번째 예 CCL19 자극 인간 차 T 림프구의 용도에 관한 것이다. 그러나 동일한 전략은 기본 마우스 T 세포, T 세포 라인 또는 케모카인 자극에 응답하여 임의의 다른 세포 유형과 함께 사용될 수있다. 여기에 제시된 게이팅 전략 포커스 이벤트 후, 단일 세포를 선택하는 일련의 윈도우를 포함한다. (그림 1) 또는 CCL19 자극 (그림 2) T 림프구 휴식의 MHC 클래스 I...

Watch this Scientific Journal Video about Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling at JoVE.com

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

케모카인 시그널링에 의해 유도 된 세포 및 분자 양극화의 신속하고 강력한 분석

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

rapid-robust-analysis-cellular-molecular-polarization-induced

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell Polarization and Migration

SCIENTISTS IN ACTION

8.0K 조회수.  Institut Cochin. Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

비디오: Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &alpha;-actinin by Confocal Imaging

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an effective invasin for human epithelial and endothelial cells. We have identified endothelial surface-located integrins as major receptors for Opc, a process which requires Opc to first bind to integrin ligands such as vitronectin and via these to the cell-expressed receptors1. This process leads to bacterial invasion of endothelial cells2. More recently, we observed an interaction of Opc with a 100kDa protein found in whole cell lysates of human cells3. We initially observed this interaction when host cell proteins separated by electrophoresis and blotted on to nitrocellulose were overlaid with Opc-expressing Nm. The interaction was direct and did not involve intermediate molecules. By mass spectrometry, we established the identity of the protein as &alpha;-actinin. As no surface expressed &alpha;-actinin was found on any of the eight cell lines examined, and as Opc interactions with endothelial cells in the presence of serum lead to bacterial entry into the target cells, we examined the possibility of the two proteins interacting intracellularly. For this, cultured human brain microvascular endothelial cells (HBMECs) were infected with Opc-expressing Nm for extended periods and the locations of internalised bacteria and &alpha;-actinin were examined by confocal microscopy. We observed time-dependent increase in colocalisation of Nm with the cytoskeletal protein, which was considerable after an eight hour period of bacterial internalisation. In addition, the use of quantitative imaging software enabled us to obtain a relative measure of the colocalisation of Nm with &alpha;-actinin and other cytoskeletal proteins. Here we present a protocol for visualisation and quantification of the colocalisation of the bacterium with intracellular proteins after bacterial entry into human endothelial cells, although the procedure is also applicable to human epithelial cells.

Visualisation and Quantification of Intracellular Interactions of Neisseria meningitidis and Human &amp;#945;-actinin by Confocal Imaging

Neisseria meningitidis (Nm), a gram negative human-specific respiratory pathogen, can bind to human &alpha;-actinin. Here we present a protocol for visualisation of colocalisation of the bacterium with intracellular &alpha;-actinin after bacterial entry into human brain microvascular endothelial cells (HBMECs).

의 세포 상호 작용 Visualisation 및 부량 Neisseria meningitidis</em공촛점 이미징에 의해 α - actinin>과 인간

The Opc protein of Neisseria meningitidis (Nm, meningococcus) is a surface-expressed integral outer membrane protein, which can act as an adhesin and an ...

연구

면역학 및 감염병학

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1 Bacterial biofilms are encased within a matrix and attached to a surface.2 Biofilm formation and development are commonly studied in the laboratory using batch systems such as microtiter plates or flow systems, such as flow-cells. These methodologies are useful for screening mutant and chemical libraries (microtiter plates)3 or growing biofilms for visualization (flow cells)4. Here we present detailed protocols for growing Staphylococcus aureus in two additional types of flow system biofilms: the drip flow biofilm reactor and the rotating disk biofilm reactor.
Drip flow biofilm reactors are designed for the study of biofilms grown under low shear conditions.5 The drip flow reactor consists of four parallel test channels, each capable of holding one standard glass microscope slide sized coupon, or a length of catheter or stint. The drip flow reactor is ideal for microsensor monitoring, general biofilm studies, biofilm cryosectioning samples, high biomass production, medical material evaluations, and indwelling medical device testing.6,7,8,9
The rotating disk reactor consists of a teflon disk containing recesses for removable coupons.10 The removable coupons can by made from any machinable material. The bottom of the rotating disk contains a bar magnet to allow disk rotation to create liquid surface shear across surface-flush coupons. The entire disk containing 18 coupons is placed in a 1000 mL glass side-arm reactor vessel. A liquid growth media is circulated through the vessel while the disk is rotated by a magnetic stirrer. The coupons are removed from the reactor vessel and then scraped to collect the biofilm sample for further study or microscopy imaging. Rotating disc reactors are designed for laboratory evaluations of biocide efficacy, biofilm removal, and performance of anti-fouling materials.9,11,12,13

The Use of Drip Flow and Rotating Disk Reactors for Staphylococcus aureus Biofilm Analysis

Protocols for utilizing open system flow biofilms with drip flow reactors and rotating disk reactors are presented in detail.

에 대한 드립 흐름 및 회전 디스크 원자로의 사용 포도상 구균 Biofilm 분석

Most microbes in nature are thought to exist as surface-associated communities in biofilms.1  Bacterial biofilms are encased within a matrix and attached ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

모기의 기능 분석을 위해 칼슘 Bioluminescence 분석 ( Aedes aegypti)와 틱 ( Rhipicephalus microplus) G 단백질 결합 수용체

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

의 이미징 HIV-1 봉투 유발 Virological 시냅스와 합성 지질 Bilayers에 신호

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

실시간, TCR의 동시 이미징 및 관련 신호 전달 단백질에 대한 TIRF 현미경 기법

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is initiated by interaction of the pathogen with intestinal epithelial cells. This interaction leads to disruption of intercellular structures such as tight junctions (TJ). TJ ensure sealing of the epithelial layer to separate host tissue from gut lumen. Recent studies provide evidence that disruption of TJ by the parasitic protein EhCPADH112 is a prerequisite for E. histolytica invasion that is accompanied by epithelial barrier dysfunction. Thus, the analysis of molecular mechanisms involved in TJ disassembly during E. histolytica invasion is of paramount importance to improve our understanding of amoebiasis pathogenesis. This article presents an easy model that allows the assessment of initial host-pathogen interactions and the parasite invasion potential. Parameters to be analyzed include transepithelial electrical resistance, interaction of EhCPADH112 with epithelial surface receptors, changes in expression and localization of epithelial junctional markers and localization of parasite molecules within epithelial cells.

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Human infection by Entamoeba histolytica leads to amoebiasis, a major cause of diarrhea in tropical countries. Infection is initiated by pathogen interactions with intestinal epithelial cells, provoking the opening of cell-cell contacts and consequently diarrhea, sometimes followed by liver infection. This article provides a model to assess the early host-pathogen interactions to improve our understanding of amoebiasis pathogenesis.

에 의해 제작 상피 손상의 분석<em&gt; Entamoeba 아메바</em&gt; 감염

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

빠른 분자 탐지 및 인플루엔자 바이러스 A와 B의 차별화

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

해 다중 단백질 신호 복합물 분자 보완성 친 화력 정화 (BiCAP)에 의해

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.