우리는 로얄 프린스 알프레드와 CRC와 정상적인 장 점막의 신선한 샘플 수집에 대한 콩코드 송환 병원의 해부 병리학 연구소에서 직원을 감사드립니다. 작품은 암 연구소 뉴사우스 웨일즈 Translational 프로그램 권한 부여에 의해 투자되었다.

<img src="/files/ftp_upload/3322/3322fig1.jpg" alt="그림 1" /> CRC의 수술 표본에서 살고 세포 현탁액의 준비를 위해 그림 1. 워크플로우. 1. 임상 샘플 disaggregation 모든 샘플은 프로토콜 번호 X08 - 164 아래의 정보를 동의를 얻어 로얄 프린스 알프레드 병원 (Camperdown, NSW, 호주)과 콩코드 송환 병원 (콩코드 웨스트, NSW, 호주)에서 수집되었다. <ol><li> 신선한 대장암 (CRC) 또는 adenoma의 표본, 그리고 정상적인 창자 점막 종양에서 적어도 10cm를 수집합니다. 절제술 후 최대 12 H 4에서 행크의 균형 소금 솔루션 산도 7.3 (HBSS) ° C에서 저장 샘플. </li><li> 생물 학적 안전 캐비닛 클래스 II의 모든 임상 샘플 처리, 인간의 병원체에 대한 안전 규정을 따르십시오. 이 메스 블레이드를 사용하여 배양 접시에있는 2mm 큐브에 샘플을 해부하다. </li><li> 37 60 분 가끔 부드러운 혼합 별도의 Eppendorf 튜브에 종양과 정상 조직을 품어 ° 2% (V / V) collagenase 타입 4 (워싱턴, 레이크 우드, 뉴저지, 미국)와 0.1이 포함된 RPMI 1640 배지의 동일한 볼륨 C 소 pancrease에서 % (W / V) deoxyribonuclease I (DNAse I, 시그마 - 올드 리치). </li><li> 포스 10 ML의 주사기에서 플런저를 사용하여 미세 와이어 메쉬 스트레이너를 통해 조직을 준 소화, HBSS로 세척을 통해 세포. </li><li> 셀 집계를 제거 200 μm의 50 μm의 Filcon 필터 (BD Biosciences)를 통해 세포 현탁액을 결과 전달합니다. DNA, 점액 및 세포 집계의 대부분은 filtrations이 시리즈 제거됩니다. </li><li> 5 분 20 °에서 400 XG에서 세포 정지를 원심 분리기. </li><li> 10% 디메틸 sulphoxide (DMSO)를 포함하는 열 inactivated FCS에서 Resuspend 세포 산탄은 -80 °에서 cryovials과 가게에서 천천히 냉동. 동결 과정은 샘플에서 점액을 감소하는 경향과 적혈구를 lyses. </li></ol> 2. 세포 캡처를위한 샘플 준비 <ol><li> DMSO를 씻어 HBSS 10 ML에서 37 ° 물 욕조, resuspend 세포에 신속하게 샘플을 좀 녹여. </li><li> 5 분 20 °에서 410 XG에서 세포 정지를 원심 분리기. </li><li> 뜨는을 가만히 따르다하고 HBSS 500 μL의 세포 펠렛을 resuspend. </li><li> 0.1 % (W / V) 실온에서 20 분 DNAse I로 샘플을 처리합니다. </li><li> trypan 파랑의 동일한 볼륨 각 세포 현탁액의 10 μL를 혼합하고 hemocytometer로 혼합 10 μL를로드합니다. 죽은 세포는 염료를 차지하면서 100 배 배율에서 가벼운 현미경을 사용하여, trypan 파랑 배제로 인해 명확한 표시 가능한 세포를 계산합니다. 최소 4 X 10 6 유력한 세포는 microarray에 셀 캡처가 필요합니다. </li><li> DNAse 처리 다음, 5 분 20 °에서 410 XG 10 ML HBSS와 원심 분리기의 세포 현탁액을 resuspend. </li><li> 200 μL의 최종 볼륨 RPMI 1640에서 supernatants 및 resuspend 세포 알약을 가만히 따르다. </li></ol> 3. 항체 microarray 셀 캡처 <ol><li> 인산염로 nitrocellulose 섹션을 찍기로 DotScan 항체 microarray를 축축하게하다하면 약 20 S.위한 식염수 (PBS)를 버퍼 조심스럽게 nitrocellulose 섹션을 감동 피하고, 접힌 Kimwipes와 microarray의 유리 가장자리를 닦으십시오. </li><li> 습기 챔버를 제공하기 위해 microarray 보육 트레이에 물을 추가합니다. 챔버에 microarray를 놓고 젖은 nitrocellulose 섹션에 RPMI 1640에 세포 현탁액을 피펫. 피펫은 심지어 세포의 확산하기 위해 nitrocellulose의 각 모서리에 방울. </li><li> 1 H.위한 37 ° C에서 microarrays를 품어 배양은 세포의 microarray에 대한 항체와 접촉에 정착하여 올 수 있습니다. 그들은에 착륙 항체에 해당하는 표면 항원을 표현 전지 캡처됩니다. </li><li> 부화 후, 언바운드 세포 (씻어 당 20의) 씻어 최소 15 ML PBS를 포함하는 세 골짜기로 부드럽게 수직 microarrays을 찍어. </li><li> 교차 연결하여 세포와 항체를 해결하기 위해 3.7 % (W / V) PBS에 포름 알데히드를 준비합니다. 부드럽게 microarray의 nitrocellulose 부분을 충당하기 위해 약 1 ML을 피펫. 상온 20 분 알을 품다. </li><li> 다음 딥 microarrays PBS (15 ML, 30의 각) 3 변화에 초과하는 포름 알데히드를 씻어 수 있습니다. </li><li> 가장자리와 Kimwipes있는 유리 슬라이드 다시 닦으하고 DotScan 스캐너 동안을 사용하여 microarray를 스캔, nitrocellulose 섹션 촉촉한입니다. 광학 스캔 혼합 세포 인구 예 : CRC 세포, leukocytes 및 종양의 다른 stromal 세포의 항원 표현 패턴을 제공합니다. </li></ol> 4. 멀티플렉싱 형광 <ol><li> 스캐너에서 microarray를 제거하고 차단 버퍼 (2 % W / V BSA, 2퍼센트 열 inactivated 인간 AB 묘약, PBS, pH를 7.3) 200 μL를 적용합니다. 20 분 상온에서 microarray 트레이에 품어. </li><li> multip 준비(Biolegend, 10 μL 알렉사 형석 647 - 안티 EpCAM, 20 μL Phycoerythrin - 안티 인 경우에는 3 번 CD (1/7.5 최종 희석 베크 만 보습 바로 앞에 달린 풀베는 날, Gladesville, NSW, 호주, # IM12824) : 알루미늄 호일로 덮여 Eppendorf 튜브에 솔루션을 lexing 샌디에이고, CA, 미국, 15분의 1 희석), 열 inactivated 인간 AB 혈청 (시그마 - 올드 리치, 성 힐, NSW, 호주)와 차단 버퍼 118 μL 2 μL. </li><li> microarray에서 초과 차단 버퍼를 배수하고 균일하게 확산, nitrocellulose 섹션에 멀티플렉싱 솔루션을 피펫. 상온에서 어두운 곳에 30 분 알을 품다. </li><li> 세로 15 ML 신선한 PBS의 세 골짜기로 microarray를 찍어, (30의 각). </li><li> 4 어둡고 저장소에있는 microarrays가 건조 해 ° 슬라이드 상자에 C. microarray는 형광의 손실없이 최대 3 개월 동안 어둠에 저장할 수 있습니다. </li><li> 50 설정된 해상도 태풍 FLA 9000 스캐너 (GE 헬스케어, Rydalmere, NSW, 호주)를 사용하여 건조 microarray 스캔 (532 nm의 레이저, PE에 대한 580 BP30 방출 필터를. 알렉사 647에 대한 633 NM 레이저와 670 BP30 방출 필터). microarrays은 유리 스캐너 트레이에서 아래쪽으로 직면하고있는 nitrocellulose 측면과 함께 스캔됩니다. </li><li> 17 TIFF 파일과 같은 형광 이미지를 사용하여 포토샵 설정 이미지 크기를 저장하는 것은 X 25cm와 해상도 72 픽셀 / cm합니다. 점들의 강도를 분석하는 DotScan 분석 소프트웨어로 이미지를 가져옵니다. </li><li> DotReader는 점 바인딩 패턴의 디지털 이미지를 캡처하고 8 비트 greyness 규모 (1-256 U) 각 항체 점에서 셀 바인딩의 밀도를 quantifies. 수시 아닌 특정 isotype 컨트롤 바인딩이 해당 면역 글로불린과 항체 isotypes에 대한 구속력이 값에서 빼는되었습니다. 각 microarray에 대한 점 형광 농도 100 %의 강도로 설정 밝은 점에 대한 정상화되었습니다. 신호 / 현장 강도. xml 파일 (원시 데이터)에 기록된 또는에서 막대 차트로 표시했습니다. pdf 파일 (최종 보고서) </li><li> Microarray heatsmaps 및 계층적 클러스터링은 TM4 Microarray 소프트웨어 스위트 (에서 MultiExperiment 뷰어 (MeV) 버전 4.4을 사용하여 수행되었다 <a href="http://www.tm4.org/mev.html">http://www.tm4.org/mev.html</a> ). 계층적 클러스터링은 전체 연동 분석 MeV를 사용하여 배경 - 조정 데이터에서 수행되었다. 유클리드 거리는 유사성 측정을 위해 사용되었다. 동일한 분산과 함께 2 꼬리 학생의 T - 테스트 결과의 통계적 중요성을 결정하는 데 사용되었다. </li></ol> 5. 대표 결과 : DotScan의 microarray의 결과는 중복 배열 사이의 일관성을 셀 바인딩 패턴을 표시합니다. 강력한 정렬 도트 바인딩 (CD44/CD29)는 배열의 영역에 배치 될 수있는 격자 수 있습니다. 그림 2는 최적의 세포 캡처 및 멀티플렉싱의 예를 보여줍니다. 그림 3은 세포 캡처 및 가능한 해결 방법 중에 발생한 몇 가지 일반적인 문제를 보여줍니다. microarray 셀 바인딩 결과는 1부터 256까지 greyness 규모에 표현 점 농도를 측정하여 계량하실 수 있습니다. 그림 4는 계층 클러스터링과 히트맵로 EpCAM - 알렉사 647 항체 물들일 58 수술 CRC 샘플에서 수치 데이터를 나타냅니다. 샘플의 수가 제한에도 불구하고, 동일한 단계 CRCs 같은 그룹에 클러스터하는 경향이있다. <img src="/files/ftp_upload/3322/3322fig2.jpg" alt="그림 2" /> 그림 2. 임상 대장암 종양 (호주 클리닉 - 병적 스테이징, ACP 단계 B1)의 세포 결합 패턴. 중복 microarray (설명)의 왼쪽 절반 항체의 위치를​​ 보여주는 (A) DotScan 항체 키. 상단 부분 DotScan의 백혈병 microarray의 원래 82 항체가 포함되어 있습니다. 특정 표면 항원에 해당하는 추가 정보는 40 항체는 CRC &#39;위성&#39;microarray으로 추가되었습니다, 문학에 - 규제로 발견했습니다. 아래 섹션 isotype 제어 항체 microarray에 바인딩 CRC 세포 (B) 광학 이미지로 구성되어 있습니다. T - 세포를 보여주는 (C) 인 경우에는 3 번 CD의 형광 이미지. CRC 세포를 보여주는 (D) EpCAM 형광 이미지. <img src="/files/ftp_upload/3322/3322fig3.jpg" alt="그림 3" /> 그림 3. 가난한 DotScan 결과와 가능한 해결 방법의 예. (A) 낮은 셀 바인딩, 솔루션 :; 최소화하기 위해 microarray에 부화하기 전에 샘플로 열 inactivated 인간 AB 혈청을 추가 솔루션 적어도 4x106 가능한 세포 (B) 바인딩 Isotype 컨트롤 바인딩이 아닌 특정 세포가 배열에 있는지 확인하십시오 isotype 제어 바인딩. 때때로, nitrocellulose에 세포가 아닌 특정 바인딩의 작은 금액은 CRC 샘플 발생하고 훨씬 결과에 영향을주지 않습니다. (C) Nitrocellulose이 부화하는 동안 밖으로 건조, 솔루션 : 예제는 전체 nitrocellulose 섹션을 포함하고 microarray는 평평한 표면에 incubated 있는지 확인하십시오. (D) 높은 배경 유물, 솔루션 :을 보장microarray는 철저하게 다음과 부화를 씻어입니다. <img src="/files/ftp_upload/3322/3322fig4.jpg" alt="그림 4" /> 그림 4. DotScan 분석 소프트웨어는 1에서 256까지 greyness 규모 셀 바인딩 밀도를 나타내는 막대 차트를 생성. 축의 숫자는 CD 항원을 참조하십시오. κ, λ, 면역 글로불린 라이트 체인,, 시그마, 표면 면역 글로불린, DCC, 대장암 단백질의 삭제, EGFR, 표피 성장 인자 수용체, FAP, fibroblast 활성화 단백질, HLA - A, B, 기타 약어는 TCR, T - 세포 수용체 아르 A, B, C 각각 C HLA - DR, 인간 백혈구 항원 DR과, 운모, MHC 클래스 I 체인과 관련된 단백질, MMP - 14, 매트릭스 metallopeptidase 14, PIGR, 고분자 면역 글로불린 수용체, TSP - 1, thrombospondin - 1; Mabthera, humanised 안티 CD20. <a href="http://www.jove.com/files/ftp_upload/3322/3322fig4large.jpg">큰 이미지를 보려면 여기를 클릭하십시오</a> .

<ol>
	<li>Steinert, R., Buschmann, T., vander Linden, M., Fels, L. M., Lippert, H., Reymond, 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=The+role+of+proteomics+in+the+diagnosis+and+outcome+prediction+in+colorectal+cancer.">The role of proteomics in the diagnosis and outcome prediction in colorectal cancer.</a> Technol. Cancer. Res. Treat. 1, 297 (2002).</li><li>Eifel, P., Axelson, J. A., Costa, J., Crowley, J., Curran, W. J., Deshler, A., Fulton, S., Hendricks, C. B., Kemeny, M., Kornblith, A. B., Louis, T. A., Markman, M., Mayer, R., Roter, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=National+Institutes+of+Health+Consensus+Development+Conference+Statement:+adjuvant+therapy+for+breast+cancer.">National Institutes of Health Consensus Development Conference Statement: adjuvant therapy for breast cancer.</a> J. Natl. Canc. Inst. 93, 979 (2001).</li><li>Swerdlow, S. H., Campo, E., Harris, H. L., Jaffe, E. S., Pileri, S. A., Stein, H., Thiele, J., Vardiman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=WHO+classification+of+tumour+of+haematopoietic+and+lymphoid+tissues.">WHO classification of tumour of haematopoietic and lymphoid tissues.</a> IARC WHO Classification of Tumours. 2, (2008).</li><li>Xiao, G. G., Recker, R. R., Deng, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+proteomics+and+cancer+biomarker+discovery.">Recent advances in proteomics and cancer biomarker discovery.</a> Clin. Med. Oncol. , (2008).</li><li>Belov, L., Mulligan, S. P., Barber, N., Woolfson, A., Scott, M., Stoner, K., Chrisp, J. S., Sewell, W. A., Bradstock, K. F., Bandall, L., Pascovici, D. S., Thomas, M., Erber, W., Huang, 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=Analysis+of+human+leukaemias+and+lymphomas+using+extensive+immunophenotypes+from+an+antibody+microarray.">Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray.</a> Br. J. Haematol. 135, 184 (2006).</li><li>Belov, L., Huang, P., Barber, N., Mulligan, S. P., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+repertories+of+surface+antigens+on+leukemias+using+an+antibody+microarray.">Identification of repertories of surface antigens on leukemias using an antibody microarray.</a> Proteomics. 3, 2147 (2003).</li><li>Zhou, J., Belov, L., Huang, P. Y., Shin, J., Solomon, M. J., Chapuis, P. H., Bokey, L., Chan, C., Clarke, C., Clarke, S. J., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+antigen+profiling+of+colorectal+cancer+using+antibody+microarrays+with+fluorescence+multiplexing.">Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing.</a> J. Immunol. Methods. 355 (1-2), 40-51 (2010).</li><li>Ellmark, P., Belov, L., Huang, P., Lee, C. S., Solomon, M. J., Morgan, D. K., Christopherson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+surface+molecules+on+colorectal+cancers.">Multiplex detection of surface molecules on colorectal cancers.</a> Proteomics. 6, 1791 (2006).</li><li>Pearson, J. P., Allen, A., Hutton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rheology+of+mucin.">Rheology of mucin.</a> Methods Mol. Biol. 125, 99 (2000).</li><li>Yang, Y. H., Dudoit, S., Luu, P., Lin, D. M., Peng, V., Ngai, J., Speed, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normalization+for+cDNA+microarray+data:+a+robust+composite+method+addressing+single+and+multiple+slide+systematic+variation.">Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.</a> Nucleic Acids Res. 30, 15 (2002).</li><li>Al Saeed, ., 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=TMA:+A+free,+open-source+system+for+microarray+data+management+and+analysis.">TMA: A free, open-source system for microarray data management and analysis.</a> BioTechniques. 34, 374-378 (2003).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 비디오에서는, 우리는 DotScan 항체 microarray는 CRC 조직의 세포 집단을위한 표면 항원 프로필을 연구하기위한 간단한 세미 양적 방법으로 사용할 수있는 방법을 보여줍니다. 에너지 종속 프로세스 (예 : 항원이 상한 및 / 또는 pseudopodia 형성) 보육시 항체 점들에 전체 세포의 회사 바인딩에 필요한 것으로 나타납니다 때문에 조직에서 가능한 단일 세포 현탁액 구하기, 실험의 ?...

The author's email has been corrected in the publication of <a href="http://www.jove.com/video/3322">Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing</a>. There was an error with the author, Jerry Zhou's, email. The author's email has been updated to:
j.zhou@uws.edu.au
from:
jzho7551@mail.usyd.edu.au

Erratum: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

<table border="1"><tbody><tr><th> 시약이나 장비의 명칭 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 </th></tr><tr><td> 행크스 &#39;균형 소금 솔루션 </td><td> 시그마 - 올드 리치 </td><td> H6136 - 10X1L </td><td> 25 MM Hepes (시그마 # H3375)와 버퍼 </td></tr><tr><td> Airpure 생물 학적 안전 캐비닛 클래스 II </td><td> 웨스팅 하우스 </td><td> 1687-2340/612 </td><td></td></tr><tr><td> 외과 블레이드 </td><td> 리빙스톤 </td><td> 090609 </td><td> 100 팩 </td></tr><tr><td> 이 MM Hepes와 RPMI 1640 </td><td> 시그마 - 올드 리치 </td><td> R4130 - 10X1L </td><td></td></tr><tr><td> Collagenase 유형 4 </td><td> 워싱턴 </td><td> 4188 </td><td></td></tr><tr><td> Deoxyribonuclease 1 </td><td> 시그마 - 올드 리치 </td><td> DN25 - 1G </td><td></td></tr><tr><td> Terumo 주사기 (10 ML) </td><td> Terumo </td><td> SS 10 L </td><td> 100 박스 </td></tr><tr><td> Filcon 필터 (200 μm의) </td><td> BD Biosciences </td><td> 340615 </td><td></td></tr><tr><td> Filcon 필터 (50 μm의) </td><td> Filcon 필터 (50 μm의) </td><td> Filcon 필터 (50 μm의) Filcon 필터 (50 μm의) 340603 </td><td></td></tr><tr><td> 태아 송아지 혈청 </td><td> Gibco / Invitrogen </td><td> 10099-141 </td><td></td></tr><tr><td> 5810 R을 원심 분리기 </td><td> Eppendorf </td><td> 7017 </td><td></td></tr><tr><td> 디메틸 sulphoxide </td><td> 시그마 - 올드 리치 </td><td> D2650 </td><td></td></tr><tr><td> Trypan 파랑 </td><td> 시그마 - 올드 리치 </td><td> T8154 </td><td></td></tr><tr><td> Hemocymeter Technocolor Neubar </td><td> Hirschmann </td><td> 사용할 수 없습니다 </td><td></td></tr><tr><td> 라이트 현미경 </td><td> 니콘 </td><td> 니콘 TMS </td><td></td></tr><tr><td> Cyrovial 튜브 </td><td> Greiner 바이오 - 원 </td><td> 121278 </td><td></td></tr><tr><td> Cryo의 동결 contrainer </td><td> Nalgene </td><td> 5100-0001 </td><td></td></tr><tr><td> DotScan 항체 microarray 키트 </td><td> Medsaic </td><td> 사용할 수 없습니다 </td><td></td></tr><tr><td> DotScan microarray 페인트 트레이 </td><td> Medsaic </td><td> 사용할 수 없습니다 </td><td></td></tr><tr><td> KimWipes </td><td> 킴벌리 - 클락 </td><td> 4103 </td><td></td></tr><tr><td> 37% 포름 알데히드 </td><td> 시그마 - 올드 리치 </td><td> F1635 - 500ML </td><td></td></tr><tr><td> DotReaderTM </td><td> Medsaic </td><td> 사용할 수 없습니다 </td><td></td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 - 올드 리치 </td><td> A9418 - 10G </td><td></td></tr><tr><td> 열 inactivated AB 혈청 2% </td><td> Invitrogen </td><td> 34005100 </td><td></td></tr><tr><td> Phycoerythrin - 복합 인 경우에는 3 번 CD에 들어 </td><td> 베크 만 보습 바로 앞에 달린 풀베는 날 </td><td> ET386 </td><td></td></tr><tr><td> AlexaFluor647 - 복합 EpCAM </td><td> BioLegend </td><td> 324212 </td><td></td></tr><tr><td> 태풍 FLA 9000 </td><td> GE 헬스케어 </td><td> 28-9558-08 </td><td> 532 NM 레이저, PE에 대한 580 BP30 방출 필터. 633 NM 레이저와 Alexa647에 대한 670 BP30 방출 필터 </td></tr><tr><td> MultiExperiment 뷰어 v4.4 </td><td> TM4 Microarray 소프트웨어 스위트 </td><td> 오픈 - 소스 소프트웨어 (참조 11) </td><td></td></tr></tbody></table>

colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing

CRC의 현재 예측과 분류 histopathologic 및 임상 결과를 통합하는 시스템을 준비에 의존합니다. 그러나, CRC 케이스의 대다수에서, 세포 기능 장애는 단백질 표현과 사후 translational 수정 수정 1 수많은 돌연변이의 결과이다. 차별화 (CD) 항원의 클러스터를 포함하여 세포 표면 항원,의 숫자 CRC 잠재적인 전조 또는 전이성 생체으로 확인되었습니다. 이러한 항원들은 종종 자신의 표현으로 종양의 진행이나 ​​다른 세포 유형, 같은과 상호 작용과 변화를 이상적인 생체를 종양 - 침투 lymphocytes (TILs)와 종양 - 관련 macrophages (리버 탐)를. 암 하위 분류와 예지를위한 immunohistochemistry (IHC)의 사용은 물론 일부 종양 유형 2,3에 대해 설정됩니다. 그러나, 하나의 &#39;표지&#39;는 clinico - 병적인 준비 이상의 전조 중요성을 표시하거나 모든 CRC 케이스의 일상 병리보고에 사용하기 위해 광범위한 수용을 받고있다. 질병 phenotypes의 전조 충화에 더 최근의 접근 방법은 여러 &#39;표시&#39;를 사용하는 표면 단백질 프로파일에 의존합니다. 같은 iTRAQ로 proteomic 기술을 이용하여 종양의 표현 프로 파일링이 biomarkers4의 발견을위한 강력한 도구이지만, 그것은 진단 실험실에서 일상적인 사용을위한 최적되지 않고 혼합 인구의 서로 다른 세포 유형을 구분할 수 없습니다. 또한, 종양 조직의 대량는 이러한 방법으로 정화 플라즈마 막 glycoproteins의 프로파일이 필요합니다. 이 비디오에서 우리는 DotScan CRC의 항체 microarray를 사용하여 disaggregated CRC 샘플에서 가능한 세포의 표면 프로테옴 프로 파일링위한 간단한 방법을 설명했다. 122 - 항체 microarray는 CRC 40 잠재적인 전조 마커의 검출을위한 위성 지역과 함께 혈통 특정 백혈구 마커, 접착 분자, 수용체 및 염증 및 면역 반응 5 마커의 범위를 인식 표준 82 - 항체 영역으로 구성되어 있습니다 . 세포는 그들이 해당 항원을 표현하는 항체에 캡처됩니다. 광학 스캔에 의해 결정 점 당 세포 밀도, 그 항원, 항체 6 항원과 친화력의 표현의 수준을 표현하는 세포의 비율을 반영합니다. CRC 조직이나 정상적인 창자 점막 들어, 광학 스캔은 세포의 혼합 인구의 immunophenotype을 반영합니다. 형광 멀티플렉싱은 다음 배열에 캡처 관심 세포의 선택된 하위 인구 프로파일에 사용할 수 있습니다. 예를 들어, 알렉사 647 - 안티 상피 세포 유착 분자 (EpCAM, CD326)는 Phycoerythrin - 안티 인 경우에는 3 번 CD에 들어, 사용하는 동안, CRC 세포뿐만 아니라 정상적인 창자 점막의 상피 세포를 감지하는 데 사용한 냄비 - 상피 분화 항원이다 T - 세포 7 침투 감지합니다. DotScan CRC microarray는 해부학적인 몸의 구조 기반 CRC의 준비 시스템 진단 대안에 대한 프로토 타입을해야합니다.

Watch this Scientific Journal Video about Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing at JoVE.com

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

그러면 표면 항원 (DotScan CRC microarray)를 인식 맞춤 항체 microarrays에 캡처되어 가능한 단일 세포를 생산하는 대장암 (CRC)의 disaggregation에 대한 절차를 설명했다. microarray에 바인딩된 세포의 하위 인구는 형​​광 염료와 태그 단클론 항체를 사용하여 형광 멀티플렉싱하여 프로파일 수 있습니다.

항체 Microarray 및 형광 멀티플렉싱을 사용하여 대장암 세포 표면 단백질 프로파일

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

colorectal-cancer-cell-surface-protein-profiling-using-an-antibody

Research

JoVE Journal

Immunology and Infection

13.9K 조회수.  University of Sydney. 그러면 표면 항원 (DotScan CRC microarray)를 인식 맞춤 항체 microarrays에 캡처되어 가능한 단일 세포를 생산하는 대장암 (CRC)의 disaggregation에 대한 절차를 설명했다. microarray에 바인딩된 세포의 하위 인구는 형​​광 염료와 태그 단클론 항체를 사용하여 형광 멀티플렉싱하여 프로파일 수 있습니다.

비디오: Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

바이러스성 병원균에 대한 임상 샘플을 화면에 판 - 바이러스성 Microarray 분석 (Virochip)를 사용

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

연구

면역학 및 감염병학

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. 
Methods for fractionating leptospiral membranes 2-5 may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids 6, 7. In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic &beta;-sheets arranged in a barrel-like structure 8, 9. In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane.
Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL5410 are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm.
The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended 10.

An efficient method to assess surface-exposure of leptospiral proteins is described. The method is specifically designed to avoid disruption of the fragile outer membrane of leptospiral cells. This technique requires employment of several negative controls to assess the integrity of the outer membrane and specificity of antibody reaction.

Leptospiral 표면 노출 단백질의 단백질 형광 분석

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

의 단일 세포 분석 바실러스 subtilis</em형광 현미경 및 유동세포계측법 사용> Biofilms

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Visualization of Bacterial Toxin Induced Responses Using Live Cell Fluorescence Microscopy

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external environment. The cell can either recover from this insult, which requires active membrane repair processes, or else die depending on the amount of toxin exposure and cell type1. In addition, these toxins induce strong inflammatory responses in infected hosts through activation of immune cells, including macrophages, which produce an array of pro-inflammatory cytokines2. Many Gram positive bacteria produce cholesterol binding toxins which have been shown to contribute to their virulence through largely uncharacterized mechanisms.
Morphologic changes in the plasma membrane of cells exposed to these toxins include their sequestration into cholesterol-enriched surface protrusions, which can be shed into the extracellular space, suggesting an intrinsic cellular defense mechanism3,4. This process occurs on all cells in the absence of metabolic activity, and can be visualized using EM after chemical fixation4. In immune cells such as macrophages that mediate inflammation in response to toxin exposure, induced membrane vesicles are suggested to contain cytokines of the IL-1 family and may be responsible both for shedding toxin and disseminating these pro-inflammatory cytokines5,6,7. A link between IL-1&beta; release and a specific type of cell death, termed pyroptosis has been suggested, as both are caspase-1 dependent processes8. To sort out the complexities of this macrophage response, which includes toxin binding, shedding of membrane vesicles, cytokine release, and potentially cell death, we have developed labeling techniques and fluorescence microscopy methods that allow for real time visualization of toxin-cell interactions, including measurements of dysfunction and death (Figure 1). Use of live cell imaging is necessary due to limitations in other techniques. Biochemical approaches cannot resolve effects occurring in individual cells, while flow cytometry does not offer high resolution, real-time visualization of individual cells. The methods described here can be applied to kinetic analysis of responses induced by other stimuli involving complex phenotypic changes in cells.

Methods for purifying the cholesterol binding toxin streptolysin O from recombinant E. coli and visualization of toxin binding to live eukaryotic cells are described. Localized delivery of toxin induces rapid and complex changes in targeted cells revealing novel aspects of toxin biology.

라이브 세포 형광 현미경을 사용하여 박테리아 독소 유도 응답의 시각화

Bacterial toxins bind to cholesterol in membranes, forming pores that allow for leakage of cellular contents and influx of materials from the external ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

호스트 병원체 단백질 - 단백질 상호 작용의 풍경의 특성을 비교 접근

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

형광 염료와 항체 라벨은 자기 단백질 A와 단백질 G 비즈를 사용하여

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in human sera and in separate preparations of polyclonal intravenous immunoglobulin (IVIg). The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure, and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. We show how protein microarray can be used to determine a combination of isotype (IgG, IgA, IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), a precursor form of a B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). During the experiment, microarrays are probed with sera from individuals with C. difficile infection (CDI), individuals with cystic fibrosis (CF) without diarrhea, healthy controls (HC), and from individuals pre- and post-IVIg therapy for the treatment of CDI, combined immunodeficiency disorder, and chronic inflammatory demyelinating polyradiculopathy. We encounter significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels between patient groups, commercial preparations of IVIg, and sera before and following IVIg administration. Also, there is a significant correlation between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C.difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, we anticipate that microarray technology may help optimize and select the most clinically useful immunotherapies for C. difficile infection in a patient-specific manner.

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

This article describes a simple protein microarray method for profiling humoral immune responses to a 7-plex panel of highly purified Clostridium difficile antigens in human sera. The protocol can be extended for the determination of specific antibody responses in preparations of polyclonal intravenous immunoglobulin.

항 원 특정 항 체 응답 다음 Clostridium 남과 어울리지 않 는 감염의 혈 청 학적인 결정에 대 한 단백질 Microarray 분석 결과

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

단일 셀 정량 mRNA와 유인원 면역 결핍 바이러스에 감염 된 CD4에 표면 단백질 표정+ T 세포 격리에서 붉은 털 원숭이

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

This manuscript describes a simple and reproducible method to perform a phagocytosis assay. The first part of this method involves building a pET-SUMO-EGFP vector (SUMO = small ubiquitin-like modifier) and expressing enhanced green fluorescence protein (EGFP) in Escherichia coli (BL21DE). EGFP-expressing E. coli is coincubated with macrophages for 1 h at 37 &#176;C; the negative control group is incubated on ice for the same amount of time. Then, the macrophages are ready for assessment. The advantages of this technique include its simple and straightforward steps, and phagocytosis can be measured by both flow cytometer and fluorescence microscope. The EGFP-expressing E. coli are stable and display a strong fluorescence signal even after the macrophages are fixed with paraformaldehyde. This method is not only suitable for the assessment of macrophage cell lines or primary macrophages in vitro but also suitable for the evaluation of granulocyte and monocyte phagocytosis in peripheral blood mononuclear cells. The results show that the phagocytic capability of peritoneal macrophages from young (eight-week-old) mice is higher than that of macrophages from aged (16-month-old) mice. In summary, this method measures macrophage phagocytosis and is suitable for studying the innate immune system function.

Using Enhanced Green Fluorescence Protein-expressing Escherichia Coli to Assess Mouse Peritoneal Macrophage Phagocytosis

Here, we present a protocol to assess mouse peritoneal macrophage phagocytosis using enhanced green fluorescence protein-expressing Escherichia coli.