우리는 USF의 유동세포계측법 핵심 시설을 인정합니다. 우리는 자원을 공유 닥터 드니즈 쿠퍼 감사드립니다. 우리는 또한 설정이 비디오의 촬영에 그들의 도움 마야 코헨, 로라 비롯와 다이애나 Latour 감사드립니다. NN은 박사 원정대 HRD # 0,929,435에 NSF FG-LSAMP 브리지에 의해 지원. 이 작품은 TG에게 수여 미국 암 학회 기관 연구 그랜트 # 93-032-13/Moffitt 암 센터에 의해 재정 지원되었다.

이전 시작하기 전 다음과 같은 솔루션을 준비할 : 3퍼센트 염색법 미디어 (SM) : 1X 인산 버퍼 염분의 -3 % 태아 소 혈청 (FBS) (PBS) 맥에서 버퍼 (MB) : - 1XPBS에서 소 혈청 (BSA)에서 0.5 % 알부민 1. 마우스에서 수확 Spleens <ol><li> (; TB 종양 베어링) Subcutaneously 1.5 X Panc02 10 5 murine 세포를 100 μl 1X PBS에서 정지로 연령 C57BL 6-8 주 / 6 마우스 (할란)을 주입. 컨트롤 마우스는 (순진) 100 μl 1XPBS를 받게됩니다. </li><li> 약 사주 포스트 분사, 이산화탄소 질식사로 쥐를 안락사시켜야. </li><li> 집게와 가위를 사용 무딘 절개로 생쥐에서 수확 spleens 그런 다음 균형을 사용해 무게. 별도의 장소 spleens는 1 X PBS를 포함하는 50 ML 원뿔 튜브를 분류. </li></ol> 2. 단일 셀 Suspensio 생성Spleens에서 Leukocytes의 N 모든 절차는 생물 안전 후드와 얼음에 보관 세포와 항체 하에서 무균 환경에서 수행되어야합니다. <ol><li> 하단쪽으로 컵의 개통으로 메쉬 스크린을 삽입하여 세포 분리 체를 조립. 그런 다음 홈 붙이 측면과 함께 스레드 영역으로 유지 링을 삽입하기 때문에 장소에서 화면을 잡고 유지 링을 강화하기 위해 고리 키를 사용합니다. 10 ML 1 X PBS가 들어있는 배양 접시에있는 조립 체를 놓습니다. </li><li> 풀 spleens 및 사용 유리 유봉은 세포 분열 체의 메쉬 스크린 반대하고 페트리 접시에 spleens를 갈아서합니다. 생쥐의 각 치료 그룹에 대해 반복합니다. </li><li> 70 μm의 셀 스트레이너와 5 ML serological 피펫을 사용하여 50 ML 원뿔 튜브로 필터 세포 현탁액. 5 분 동안 12,000 RPM (250-300 XG)에서 원심 분리기. </li><li> 비장 당 5 ML 1 X RBC의 용해 버퍼에 뜨는 및 resuspend 펠릿 제거. Pipet 위아래로 풍성히. 5 분 동안 상온에서 품어. 1 X PBS의 20 ML을 추가하여 반응을 중지합니다. Pipet 위아래로 풍성히. 5 분 동안 12,000 RPM (250-300 XG)에서 원심 분리기. </li><li> 20 ML 멸균 1 X PBS에 뜨는 및 resuspend 펠렛을 제거합니다. Pipet 위아래로 풍성히. </li><li> 50 μl가 5x10 5-1x10 6 전지 (- 2x10 7 셀 / ML 1x10 7 세포 / ml) 쇼핑하는 것과 같습니다 있도록 trypan 파랑, 3 % SM에서 원하는 농도에서 hemacytometer과 resuspend를 사용하여 세포를 세어보세요. </li></ol> 3. 유동세포계측법를 사용 MDSC의 세포 - 표면 염색법 / Immunophenotyping <ol><li> (; 맥-1-FITC, GR-1-APC 및 DAPI 얼룩 없음, NS) 제어 및 보정 컨트롤에 대한 실험 시료와 단일 얼룩을위한 96 - 웰 V 바닥 플레이트의 우물을 레이블. </li><li> 96도 V-맨 아래 판에서 / 잘 각각의 우물에 splenocytes 50 μl에 상응 5x10 5-1x10 6 셀을 추가합니다. 원심 분리기5 분 용 12,000 RPM (250-300 XG)에서 판. </li><li> 얼음 1.5 ML의 microcentrifuge 튜브에 3 % SM에서 희석 마우스 BD FC 블럭 (쥐 안티 마우스 CD16/32 단클론 항체)의 &quot;마스터 믹스&quot;(MM)를 준비합니다. 출발점으로서, 당 음 50 μl의 최종 볼륨의 3 % SM 1 μg FC 블록을 사용합니다. </li><li> 조심스럽게 세포 펠렛의 중단없이 폐기물 컨테이너 또는 싱크대에 다시 걸쳐 빠르게 반전 판으로 96도 V-맨 아래 접시의 각 우물에서 뜨는을 제거합니다. </li><li> 와동, 짧게 5 초 동안 FC 블록 MM을 원심 분리기와 96도 V-맨 아래 판의 모든 산탄 50 μl를 추가합니다. 부드럽게 자신의 우물에 샘플을두고, 아래로 pipetting 및하여 잘 섞는다. 얼음 어둠 속에서 15 분 동안 접시를 품어. 5 분 동안 12,000 RPM (250-300 XG)에서 접시를 원심 분리기. </li><li> 샘플 FC 블록으로 품어 동안 얼음 1.5ml microcentrifuge 튜브의 3 % SM에서 희석 fluorochrome-복합 항체의 &quot;마스터 믹스&quot;(MM)를 준비합니다. 항체가 titrated해야절차를 더럽히는 것을위한 최적의 dilutions를 확인하십시오. 출발점으로서, 이런 샘플 당 50 μl의 최종 볼륨의 3 % SM에서 1시 25분 맥-1-FITC의 희석 및 GR-1-APC의 1시 20분 희석을 결합. </li><li> 이전에 설명한대로 조심스럽게 96도 V-하단에 각 우물에서 뜨는 제거합니다. </li><li> 와동은 짧게 5 초 동안 형광등-복합 염색법의 항체의 MM을 원심 분리기 및 제어 및 실험 쥐똥 50 μl를 추가합니다. 부드럽게 최대 pipetting 및 아래쪽으로 잘 섞는다. 단일 얼룩 보상 내용 잘 당 50 μl의 최종 볼륨의 3 % SM에서 맥-1-FITC의 1시 25분 희석, GR-1-APC 75 NG / DAPI의 ML의 1시 20분 희석을 추가 각각 웰스 있습니다. (얼룩 없음) 잘 흠없는 50 μl 3 % SM을 추가합니다. 잘 섞어서 얼음에 어두운 30 분 96도 V-맨 아래 판에서 세포를 품어. </li><li> 라벨 FACS 튜브는 (5 ML, 12mm X 75mm의 폴리스티렌 원형 바닥 튜브) 96 - 웰 V-하단 플레이트의 각도에 대응합니다. 각 FACS 200 μl 3 % SM 추가튜브. </li><li> 5 분 동안 12,000 RPM (250-300 XG)에서 접시를 원심 분리기와 supernatants를 제거합니다. 각 펠렛 100 μl 3 % SM을 추가하여 알약을 씻어 부드럽게 최대 pipetting 및 아래쪽으로 잘 섞는다. 5 분 동안 12,000 RPM (250-300 XG)에서 접시를 원심 분리기. 한 번 더 씻어 단계를 반복합니다. </li><li> 이전에 설명한대로 조심스럽게 96도 V-하단에 각 우물에서 뜨는 제거합니다. 100 μl 3 % SM에서 각각의 펠렛을 Resuspend 잘 섞는다. </li><li> 그들의 각각 분류 FACS 관으로 96도 V-맨 아래 접시의 각 우물에서 100 μl resuspended 펠렛을 전송합니다. </li><li> cytometry 분석을 섞여 이전하기 위해, 75 NG / ML DAPI 제어 및 실험 샘플 및 DAPI 단일 얼룩 보상 컨트롤을 추가합니다. </li><li> MDSC 비율의 흐름 cytometric 데이터 수집을 수행합니다. 부정적인 (아무 얼룩 제어)와 하나의 긍정적인 컨트롤을 사용하여 보정을 수행합니다. 있도록 로그 스케일에서 앞으로 (FSC) 대 측면 산란 (SSC)를 표시하는 도트 음모를 설정의 백혈구 인구관심을 확인할 수 있습니다. 파편과 낮은 순방향 및 사이드 흩어있는 대단히 짧은 시간을 제외한 모든 leukocytes에 큰 성문을 그립니다. 이 부모 게이트에서 DAPI-(라이브) 세포에 SSC 대 DAPI와 게이트를 표시 새로 도트 음모를 만듭니다. 이 새로 문이 인구를 선택하고 더블 긍정 맥-1 대 GR-1과 게이트를 표시하는 도트 줄거리 만듭니다 (맥-1 + GR-1 +) MDSC. </li></ol> 4. GR-1 + Leukocytes의 자성 농축 <ol><li> 5 분 용 12,000 RPM (250-300 XG)에서 적절하게 라벨이 FACS 튜브 및 원심 분리기로 나누어지는 1x10 7 남아있는 흠없는 leukocytes. </li><li> 1.5 ML의 microcentrifuge 튜브 GR-1-PE 항체의 MM을 준비합니다. 최대 10 7 세포에 대한 내용은 샘플 당 50 μl 메가바이트에 GR-1-PE 항체의 1시 10분 희석을 사용합니다. 큰 세포 숫자의 경우 적절하게 볼륨을 잡고. 간단히, 5 초 동안 원심 분리기 FACS 튜브의 leukocytes에 추가 및 4시 15 분 ° 어둠 속에서 C에 대해 품어. </리&gt; <li> 5 분 용 12,000 RPM (250-300 XG)에 FACS 튜브, 원심 분리기에 MB 중 두 ML을 추가하고 뜨는 버리고. </li><li> 1.5 ML의 microcentrifuge 튜브에 안티 - 체육 MicroBeads의 MM을 준비합니다. 최대 10 7 세포에 대한 내용은 샘플 당 200 μl 메가바이트에 방지 PE MicroBeads의 1시 4분 희석을 사용합니다. 큰 세포 숫자의 경우 적절하게 볼륨을 잡고. 간단히, 5 초 동안 원심 분리기 FACS 튜브의 leukocytes에 추가 및 4시 15 분 ° 어둠 속에서 C에 대해 품어. </li><li> 5 분 용 12,000 RPM (250-300 XG)에 FACS 튜브, 원심 분리기에 MB 중 두 ML을 추가하고 뜨는 버리고. SB 3 ML에 Resuspend 펠릿. 새로운 라벨 50 ML 원뿔 튜브로 70 μm의의 스트레이너를 통해 필터링합니다. </li><li> 준비 및 주요 자동차 맥 프로 구분 기호. 적절한 솔루션과 비어있는 폐기물 용기와 리필은 모든 병, 필요한 경우. 악기에 관한 켜고 유체 용기 및 초기화 후 열 (들)의 상태를 검사합니다. 모든 기호가 녹색이어야합니다. 메뉴 상단에서 &quot;분리&quot;를 선택다음 메뉴 표시줄은 아래쪽 메뉴 표시줄에서 &quot;지금 씻으십시오.&quot; 마중물 프로세스를 시작하려면 &quot;실​​행&quot;다음에 팝업 옵션에서 &quot;린스&quot;를 선택하십시오. 마중물 프로세스가 성공적으로 완료되면 악기 그러면 그것 상태 메뉴에서 &quot;분리 준비&quot;이라고 표시됩니다. </li><li> 행 B의 부정적인 분획 수집 및 행 C. 긍정적인 분수 컬렉션 15 ML 원뿔 관을위한 행, 50 ML 원뿔 튜브의 자기 (磁 气)라고 표시된 셀로 튜브 크기와 장소에 적합한 냉장 튜브 랙 50 ML 원뿔 튜브를 선택하세요 </li><li> 샘플에서 민감한 모드에서 표시된 타겟 세포의 긍정적인 선택에 대해 &quot;POSSEL_S&quot;세포 분리 프로그램을 선택하십시오. 자기 (磁 气)라고 표시된 대상 세포 automats 칼럼에 보관되며, 레이블이 지정되지 않은 세포를 자석의 자동 철회에 행 B.에 부정적인 분수 수집 관으로 출시되며, 표시된 대상 세포는 튜브의 행 C에서 긍정적인 컬렉션 튜브에 발표 예정 랙. </li></ol>+ 농축 Leukocytes의 포스트 정렬 분석 <ol><li> GR-1을 재계산 +와 GR-1 - trypan 파란색 hemacytometer를 사용하여 분수. 3 % 염색법 매체에서 원하는 농도에서 Resuspend 세포가 50 μl가 5x10 5-1x10 6 전지 (1x10 7 셀 / ML - 2x10 7 세포 / ml) 쇼핑하는 것과 같습니다 있도록하고 correspondingly 분류 FACS 튜브 50 μl를 전송합니다. </li><li> 샘플 당 50 μl의 최종 볼륨 3 % SM에서 1시 25분 희석에서 유일한 맥-1의 MM을 준비합니다. 세포를 착색하고 유동세포계측법 분석 GR-PE, 맥-1 FITC와 DAPI의 단일 얼룩 보상을 준비합니다. 200 μl 3퍼센트 SM 및 DAPI의 생존을 염색하는 등 이전에 설명을 추가합니다. </li><li> 비율 또한 전후 autoMACS 농축이 MDSC 비율을 비교하는 - GR-1 +와 GR-1을 결정하는 흐름 cytometric 데이터 수집을 수행합니다. </li></ol> 6. 대표 결과 여기에서 우리는 R을 보여<html> MDSC (그림 1)으로 분류 후속 FACS를위한 풀링, 순진 spleens에서 GR-1 + leukocytes의 autoMACS 농축을위한 epresentative 결과입니다. 1x10 6 나이브 leukocytes는 사전 autoMACS 정렬로, BD LSRII 악기를 사용하여 MDSC 비율을 식별하는 데 맥-1-FITC와 GR-1-APC의 항체 물들일되었다. 1x10 7 나이브 leukocytes 그 다음 autoMACS 프로 구분 기호를 사용하여 GR-1 + leukocytes의 농축을위한 안티 GR-1-체육 항체와 PE-MicroBeads 물들일되었다. 포스트 autoMACS 농축, GR-1의 GR-1 비율 +와 GR-1 - 수집의 분수가 유동세포계측법를 사용하여 평가되었다. 1x10 6 GR-1 + leukocytes는 유동세포계측법에 의해 종양 베어링 leukocytes를 풀링되지 않은 농축에 순진 leukocytes을 풀링된 농축의 MDSC 비율을 분석하고 비교할 맥-1-FITC 항체로 제거하고 물들어 있었다. fig1.jpg &quot;/&gt; 정렬 MDSC FACS에 대한 순진 GR-1 + leukocytes의 그림 1. AutoMACS 농축. Spleens는 췌장 종양 베어링과 순진 생쥐에서 수확 및 단일 세포 현탁액으로 처리되었다. 나이브 leukocytes 표면의 유동세포계측법 분석은 사전 autoMACS 농축 ()로, 맥-1과 GR-1 형광 - 복합 항체 물들일. 유동세포계측법의 GR-1 분석 + (B) 및 GR-1 - GR-1-체육 항체 및 안티 PE MicroBeads 물들일 풀링된 순진 leuckocytes에서 GR-1 + 세포 (C) 분수 포스트 autoMACS 농축. MDSC 및 GR-1 유동세포계측법 분석 GR-1 + 비율 후 autoMACS 농축 종양 베어링 생쥐 (E)에서 비 농축 풀링된 leukocytes (순진 마우스, N = 5에 비해 풀링된 순진 leukocytes (D)에서 + 세포 ; 종양 베어링 마우스, N = 3). MDSC 및 GR-1 비율은 대표적인 윤곽 플롯과 histograms의 문이 있습니다.

<ol>
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Immunol. 199, 273-281 (2010).</li><li>Pilon-Thomas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+Pancreatic+Adenocarcinoma+Dampens+SHIP-1+Expression+and+Alters+MDSC+Homeostasis+and+Function.">Murine Pancreatic Adenocarcinoma Dampens SHIP-1 Expression and Alters MDSC Homeostasis and Function.</a> PLoS One. 6, (2011).</li><li>Panopoulos, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT3+governs+distinct+pathways+in+emergency+granulopoiesis+and+mature+neutrophils.">STAT3 governs distinct pathways in emergency granulopoiesis and mature neutrophils.</a> Blood. 108, 3682-3690 (2006).</li><li>Preynat-Seauve, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extralymphatic+tumors+prepare+draining+lymph+nodes+to+invasion+via+a+T-cell+cross-tolerance+process.">Extralymphatic tumors prepare draining lymph nodes to invasion via a T-cell cross-tolerance process.</a> Cancer Res. 67, 5009-5016 (2007).</li><li>Davies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+separations+by+flow+cytometry.">Cell separations by flow cytometry.</a> Methods Mol. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amine+reactive+dyes:+an+effective+tool+to+discriminate+live+and+dead+cells+in+polychromatic+flow+cytometry.">Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry.</a> J. Immunol. Methods. 313, 199-208 (2006).</li><li>Safarik, I., Safarikova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+magnetic+techniques+for+the+isolation+of+cells.">Use of magnetic techniques for the isolation of cells.</a> J. Chromatogr. B. Biomed. Sci. Appl. 722, 33-53 (1999).</li><li>Collazo, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SHIP+limits+immunoregulatory+capacity+in+the+T-cell+compartment.">SHIP limits immunoregulatory capacity in the T-cell compartment.</a> Blood. 113, 2934-2944 (2009).</li><li>Mack, E., Neubauer, A., Brendel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA+yield+from+small+cell+populations+sorted+by+flow+cytometry+applying+different+isolation+procedures.">Comparison of RNA yield from small cell populations sorted by flow cytometry applying different isolation procedures.</a> Cytometry. A. 71, 404-409 (2007).</li><li>Strauss, L., Czystowska, M., Szajnik, M., Mandapathil, M., Whiteside, T. 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관심의 어떠한 충돌 선언 없습니다.

이것은 여러 동물 모델에서 다른 림프 조직에 적용 MDSC 인구를 처리하고 immunophentyping에 대한 자세한 방법입니다. 특히 autoMACS 농축은 splenocytes 4 GR-1 고갈, splenocytes과 림프절에서 5 골수양 하위 집합의 정화, CD8의 골수 neutrophils 14 분리와 정제 + 비장에서 T 세포 등 각종 백혈구 인구의 격리에 사용될 수 있습니다 및 림프절 15. 에 관계없이 관심있는 세포 인구...

<table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> # 카탈로그 </td><td> 의견 </td></tr><tr><td> 1X 인산은 염분 버퍼 </td><td> 써모 과학 Hyclone </td><td> SH30028.02 </td><td> 칼슘 2 + / MG 2 + / 페놀 레드 부담 </td></tr><tr><td> 소 혈청 (BSA)로부터 알부민 </td><td> 시그마 - 올드 리치 </td><td> A7906 </td><td> BSA는 PBS에 그대로 녹아하자, 무균 환경 </td></tr><tr><td> 태아 소 혈청 (FBS) </td><td> 써모 과학 Hyclone </td><td> SV3001403HI </td><td> Inactivated 열, 무균 환경 </td></tr><tr><td> 쥐 안티 마우스 CD16/32 단클론 항체 (FC 블록) </td><td> BD Biosciences </td><td> 553,142 </td><td> 멸균 환경 </td></tr><tr><td> 안티 마우스 CD11b (Mac 용-1) FITC </td><td> eBiosciences </td><td> 11-0112 </td><td> 불모의환경 </td></tr><tr><td> 안티 마우스 Ly6G (GR-1) APC </td><td> eBiosciences </td><td> 17-5931 </td><td> 멸균 환경 </td></tr><tr><td> 안티 마우스 Ly6G (GR-1) PE </td><td> eBiosciences </td><td> 12-5931 </td><td> 멸균 환경 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D1306 </td><td> 시리얼 희석 무균 환경 </td></tr><tr><td> 세포 분리 체 </td><td> 시그마 - 올드 리치 </td><td> CD1-1KT </td><td> 사용하기 전에 압력솥 </td></tr><tr><td> 70 μm의 제품 스트레이너 </td><td> BD Biosciences </td><td> 352,350 </td><td> 멸균 환경 </td></tr><tr><td> 1X RBC의 용해 완충액 </td><td> eBiosciences </td><td> 00-4333-57 </td><td> 무균 환경, 사용하기 전에 실온에 따뜻한 </td></tr><tr><td> 배양 접시 </td><td> 피셔 과학 </td><td> 08-757-12 </td><td> 멸균 환경 </td></tr><tr><td> 50ml 죄수ICAL 튜브 </td><td> 써모 과학 </td><td> 339,652 </td><td> 멸균 환경 </td></tr><tr><td> 5ml 12X75mm 폴리스티렌 둥근 바닥 튜브 </td><td> BD Biosciences </td><td> 352,054 </td><td> FACS 튜브로 알려진, 무균 환경 </td></tr><tr><td> 96도 V-하단 플레이트 </td><td> 코닝 </td><td> 3897 </td><td> 멸균 환경 </td></tr><tr><td> Trypan 블루 </td><td> Cellgro </td><td> 25-900-CI </td><td> 멸균 환경 </td></tr><tr><td> PE MicroBeads </td><td> Miltenyi Biotec </td><td> 130-048-801 </td><td> 멸균 환경 </td></tr><tr><td> AutoMACS 프로 구분자 </td><td> Miltenyi Biotec </td><td> 130-092-545 </td><td></td></tr><tr><td> AutoMACS 열 </td><td> Miltenyi Biotec </td><td> 130-021-101 </td><td></td></tr><tr><td> 버퍼를 실행 AutoMACS </td><td> Miltenyi Biotec </td><td> 130-091-221 </td><td&gt; </td></tr></tbody></table>

preparation of myeloid derived suppressor cells (mdsc) from naive and pancreatic tumor-bearing mice using flow cytometry and automated magnetic activated cell sorting (automacs)

MDSC이지나 macrophages, 돌기 세포 및 기생충 감염, 염증, 외상 스트레스, 그라 프트 - 대 - 숙주 질환, 당뇨병 및 암 1-7 포함한 병리 학적 조건에서 림프 장기에 축적 granulocytes의 이기종 인구입니다. 생쥐에서 MDSC 특급 맥-1 (CD11b)과 GR-1 (Ly6G 및 Ly6C) 표면 항원 7. 그것은 MDSC 잘 그들이 크게 확장되는 다양한 종양 베어링 호스트에서 공부하고 있습니다 그리고 7-10 순진 대응에 비해 안티 종양 면역 반응을 억제하는 것이 중요합니다. 그러나 병적인 상태에 따라 억제 11,12의 독특한 메커니즘과 목표가있는 MDSC의 다른 subpopulations가 없습니다. 따라서 가능한 MDSC 인구를 분리하는 효과적인 방법은 시험 관내 및 생체내의 억제 그들의 서로 다른 분자 메커니즘을 elucidating에 중요하다. 최근에는 Ghansah 그룹은 murine 췌장암 모델에서 MDSC의 확장을 보도했다. 우리의 종양 - 베어링 MDSC은 항상성의 손실을 표시하고 순진 MDSC 13에 비해 진압 기능을 향상시켰습니다. MDSC 비율은 순진 대 종양 베어링 생쥐의 림프 구획에 훨씬 적습니다. 이것은 종종 이러한 MDSC의 정확한 비교 분석을 방해 주요 경고입니다. 따라서 사전에 형광 활성 세포 정렬 (FACS)으로 순진 생쥐에서 GR-1 + leukocytes을 풍부하게하는 것은 순결, 생존을 향상하고 크게 정렬 시간을 줄여줍니다. 이들은 정렬 빠른 FACS에 대한 풍부한있다 그러나 종양 베어링 생쥐에서 GR-1 + leukocytes의 농축은 선택 사항입니다. 따라서이 프로토콜에서는, 우리는 MDSC을 immunophenotyping하고 적시에 MDSC 정렬을위한 나이브 생쥐의 spleens에서 GR-1 + leukocytes을 풍요로운의 매우 효율적인 방법을 설명합니다. 면역 적격 C57BL / 6 마​​우스는 murine Panc02 CE로 주사하고재편은 subcutaneously 순진 마우스 1XPBS을받을 반면. 약 30 일 접종을 게시할 spleens는 세포 분열 체를 사용하여 단일 세포 현탁액으로 수확과 처리됩니다. Splenocytes 그러면 적혈구 (RBC)은 lysed되며 이러한 leukocytes의 나누어지는은 유동세포계측법을 사용 immunophenotype MDSC 비율에 맥-1과 GR-1에 대해 fluorochrome - 복합 항체를 사용하여 묻은 게있다. 병렬 실험에서, 순진 생쥐에서 전체 leukocytes는 자동 마그네틱 활성화 셀 정렬 (autoMACS) 프로 구분 기호를 사용하여 PE-MicroBeads 함께 incubated하고 긍정적으로 선택된 형광등-복합 GR-1 항체, 물들일 수 있습니다. 다음으로, GR-1 + leukocytes의 나누어지는은 유동세포계측법를 사용 MDSC 비율의 증가를 식별하는 맥-1 항체 물들일 수 있습니다. 이제 이러한 Gr1 + 농축 leukocytes는 생체내 및 시험 관내의에서 (순진 대 종양 베어링) 비교 분석에 사용되는 MDSC의 분류 FACS 준비 </em&gt;를 assays.

Watch this Scientific Journal Video about Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell S

Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

이것은 골수양 유래 억제 세포 (MDSC)를 immunophenotyping 및 GR-1 풍요로운의 신속하고 포괄적인 방법입니다<sup&gt; +</sup마우스 spleens에서&gt; leukocytes. 이 방법은 실용적 GR-1에 대한 풍부 정렬 유동세포계측법 및 AutoMACS 전지를 사용<sup&gt; +</sup용도 MDSC으로 정렬 FACS로 사전&gt; leukocytes<em&gt; 생체내에서</em&gt;과<em&gt; 체외에서</em&gt; assays.

유동세포계측법 및 자동화된 마그네틱 활성화 셀 정렬 (AutoMACS)를 사용 나이브 및 췌장 종양 베어링 마우스에서 골수양 파생 억제 세포 (MDSC)의 작성

MDSC are a heterogeneous population of immature macrophages, dendritic cells and granulocytes that accumulate in lymphoid organs in pathological ...

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Medicine

Preparation of Myeloid Derived Suppressor Cells (MDSC) from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting (AutoMACS)

26.1K 조회수.  University of South Florida Morsani College of Medicine. 이것은 골수양 유래 억제 세포 (MDSC)를 immunophenotyping 및 GR-1 풍요로운의 신속하고 포괄적인 방법입니다 + leukocytes. 이 방법은 실용적 GR-1에 대한 풍부 정렬 유동세포계측법 및 AutoMACS 전지를 사용 + leukocytes 생체내에서과 체외에서 assays.

비디오: Preparation of Myeloid Derived Suppressor Cells MDSC from Naive and Pancreatic Tumor-bearing Mice using Flow Cytometry and Automated Magnetic Activated Cell Sorting AutoMACS

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

정렬 줄기 세포 Assays 및 흐름에 대한 기본 뇌 종양 조직의 처리

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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의학

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

인간 치과 펄프의 줄기 세포의 분리, 특성화 및 비교 차별화 두 가지 방법을 사용하여 영구 치아에서 파생

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

인간 혈청에있는 N-메틸-D-아스파 라진 산염 수용체 또는 도파민 2 수용체에 대한 항체를 검출하는 높은 처리량 유동 세포 계측법 세포 기반 분석

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

임상 및 전임상 연구 응용 프로그램에 대한 종양 세포 (CTC) 분석 실험을 순환 반자동의 적응

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

나노 바디 및 항체 기반의 검증<em&gt; 생체</em마우스에서&gt; 종양 이종 이식 NIRF 이미징 실험 사용<em&gt; 전의 VIVO</em&gt; 유동 세포 계측법 및 현미경

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

다중 매개 변수 형광 활성화 된 셀 정렬하여 인간의 림프 내피 세포의 분리

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

췌장암 새로운 치료 전략을 개발하기위한 전지 특성 줄기 공부

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism (Microcystis aeruginosa) Using Flow Cytometry

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 &#181;M after 30 min of incubation and the orange nucleic acid probe at 1 &#181;M after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 &#181;M concentrations and higher in both probes exhibited a type of non-specific staining, whereby &#39;live&#39; cells produced a target fluorescence, leading to an over representation of &#39;non-viable&#39; cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.

Molecular Probe Optimization to Determine Cell Mortality in a Photosynthetic Organism Microcystis aeruginosa Using Flow Cytometry

Microbial populations contain substantial cell heterogeneity, which can dictate overall behavior. Molecular probe analysis through flow cytometry can determine physiological states of cells, however its application varies between species. This study provides a protocol to accurately determine cell mortality within a cyanobacterium population, without underestimating or recording false positive results.

분자 프로브 최적화 (광합성 생물의 세포 사망을 확인하는 방법<em&gt; Microcystis aeruginosa의</em&gt;) 유동 세포 계측법을 사용하여

Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.

We describe two complementary methods using the fluorescence ubiquitination cell cycle indicator (FUCCI) and image analysis or flow cytometry to identify and isolate cells in the inner G1 arrested and outer proliferating regions of 3D spheroids.