이 작품은 WM으로 국립 보건원에서 보조금 (CA127574, CA107040 및 CA09071)에서 지원이

1. 마우스에서 수확 Xenografts <ol><li> Dulbecco의 수정된 이글 중간 (DMEM) 10 % 태아 송아지 혈청 (FCS), 페니실린 - 스트렙토 마이신, 200 U / ML Collagenase 종류 IV, 그리고 0.6 U / ML dispase와 함께 보충 : 세포 분리 버퍼를 준비합니다. </li><li> 가장 큰 지름 1cm 미만입니다 피하 인간의 췌장암의 이종 이식을 품고 athymic (뉴 + / 뉴 +) 마우스는 이산화탄소 질식 및 경추 전위에 의해 euthanized있다. </li><li> 피하 종양은 집게와 가위를 사용 무딘 절개로 마우스의 측면에서 수확하고 세포 분리 버퍼에 즉시 배치됩니다. </li><li> 종양은 무게와 종양 조직 1g 당 10 ML 세포 분리 버퍼에 다시 배치됩니다. </li></ol> 2. Xenografts에서 단일 셀 서스펜션을 생성 <ol><li> 멸균 biosafety 캐비닛에 근무, 종양 세포 분리 버퍼 1 ML과 100 X 20mm 문화 요리로 전송됩니다. 종양은 기계 살균 면도날과 집게를 사용하여 다진 있습니다. 종양 세포 현탁액은 5 ML serological pipet을 통해 10 번 triturated입니다. 종양 조각은 5 ML serological pipet을 방해할하지 않을 정도로해야합니다. </li><li> 종양 세포 현탁액이 세포 분리 버퍼의 나머지 볼륨 (50 % 미만 가득)가 포함된 50 ML 원뿔 관에 전송하고 1 분 최대 속도 vortexed입니다. </li><li> 종양 세포 현탁액은 다음 ° C 2 시간 일분 매 20 분 동안 간헐적으로 vortexing와 함께 37 incubated입니다. </li></ol> 3. 세포 조직 파편의 서스펜션과 죽은 세포를 고갈 <ol><li> 2 시간 배양 후 세포 현탁액은 1 분 최대 속도 vortexed입니다. </li><li> 세포 현탁액은 다음 70 μm의 필터를 통과하고 신선한 50 ML 원뿔 튜브의 수집 관 당 15 ML의 최종 볼륨으로 조정됩니다. </li><li> 죽은 세포 및 조직의 잔해로부터 더욱 분리 가능한 세포, 종양 세포 현탁액은 Ficoll - Paque 플러스를 사용하여 밀도 원심 분리로 구분됩니다. Ficoll - Paque 플러스의 underlayment는 플러스 천천히 두 레이어를 혼합하지 않도록주의되는 세포 현탁액 아래 Ficoll - Paque 15 ML을 pipeting에 의해 생성됩니다. </li><li> 세포 현탁액과 Ficoll 레이어는 브레이크와 함께 30 분 동안 500x g에 centrifuged집니다 것은 해제. </li><li> Ficoll - Paque 플러스 및 세포 분리 버퍼의 인터페이스에서 전지 수집 신선한 50 ML 원뿔 튜브로 전송됩니다. </li><li> 신선한 DMEM 10 % FCS와 함께 보충 것은 그것을 희석하기 위해 세포 현탁액에 추가됩니다 1시 3분 (예 : 20 ML 신선한 미디어 세포 현탁액의 10 ML에 추가). </li><li> 세포는 10 분 decanted 미디어 450x g에서 원심 분리하여 수집하고 있으며, 세포 펠렛은 ALDEFLUOR 버퍼 1 ML에 resuspended 있습니다. </li><li> 세포 현탁액의 열 microliters 10 μL trypan 파랑, 가능한 세포를 hemocytometer를 사용하여 계산됩니다와 희석 수 있습니다. 죽은 세포 또는 조직 파편 많은이 단계에서 언급하는 경우에는 다음 밀도 원심 분리하여 세포 분리는 (- 3.7 단계 3.3) 반복 수 있습니다. </li></ol> 4. 형광 활성 세포 정렬에 대한 세포를 얼룩 <ol><li> 로 분류 일곱 12 X 75mm의 폴리스티렌 테스트 튜브는 다음과 같습니다 <ol><li> 흠없는 </li><li> CD44 - APC </li><li> CD24 - PE </li><li> 마우스 CD31 - 비오틴 + 마우스 혈통 - 비오틴 + 마우스 H - 2Kd - 비오틴 </li><li> ALDEFLUOR </li><li> ALDEFLUOR + DEAB + IgG 2bκ - APC + IgG 2aκ - PE </li><li> ALDEFLUOR + CD44 - APC + CD24 - PE + 마우스 CD31 - 비오틴 + 마우스 혈통 - 비오틴 + 마우스 H - 2Kd - 비오틴. </li></ol></li><li> 만 5-10 셀 / ML의 최종 농도 ALDEFLUOR 버퍼 2 ML에 세포 현탁액을 희석하여 얼음 계속. 튜브 # 1, 2, 3, 4에 세포 현탁액을 100 μL를 추가하고 얼음에 있습니다. 튜브 # 6-1 μL DEAB를 추가하고 얼음 계속. 1000 희석 나머지 세포 현탁액에 ALDEFLUOR 시약 추가 - 접어 (예 : 1.6 ML의 세포 현탁액을 1.6 μL ALDEFLUOR 시약 추가) 잘 혼합하고, 얼음에 놓으십시오. 튜브 # 7-100 튜브 # 5와 6이 혼합물 μL, 나머지 혼합물 (1.4 ML)를 전송합니다. 40 분 37 ° C 물 목욕에있는 튜브를 모두 품어. 튜브의 바닥에 정착에서 세포를 방지하기 위해 세포 현탁액마다 10 분 정도 섞는다. </li><li> 450x g에서 세포와 4 원심 분리기 ° C 10 분, 다음 버퍼를 가만히 따르다하십시오. 100 μL ALDEFLUOR 버퍼에 튜브 # 1 5 알약을 Resuspend. 튜브 # 2, 3, 4, 6하려면 다음과 같이 희석 적절한 항체를 포함하는 ALDEFLUOR 버퍼 100 μL를 추가 : IgG 2bΚ - APC, IgG 2aΚ - PE, CD44 - APC, 그리고 CD24 - PE에 대한 1시 20분항체, 마우스 CD31 - 비오틴에 대한 1:100, 마우스 혈통 - 비오틴, 마우스 H - 2Kd - 비오틴 항체. 튜브 위해 # 7은 1.4 CD44 - APC의 1시 20분 희석을 포함 ALDEFLUOR 버퍼의 ML, 그리고 CD24 - PE 항체와 마우스의 1:100 희석 CD31 - 비오틴을 추가, 마우스 혈통 - 비오틴, 마우스 H - 2Kd - 비오틴 항체. 10 분 얼음 셀 suspensions을 품어. </li><li> 450x g에서 세포와 4 원심 분리기 ° C 10 분, 다음 버퍼를 가만히 따르다하십시오. 튜브 # 1, 2 산탄, 3, 5, 100 μL ALDEFLUOR 버퍼에서 6 Resuspend. streptavidin - PerCP 단백질의 1:100 희석을 포함 ALDEFLUOR 버퍼의 1.5 ML를 준비합니다. 튜브 # 4-100 μL를 추가하고 튜브 # 7-1.4 ML를 추가합니다. 10 분 얼음 셀 suspensions을 품어. </li><li> 450x g에서 세포와 4 원심 분리기 ° C 10 분, 다음 버퍼를 가만히 따르다하십시오. 튜브 # 1, 2 산탄, 3, 4, 5, 200 μL ALDEFLUOR 버퍼에서 6 Resuspend. 2 μg / ML의 propidium 요오드화물을 포함하는 제품 ALDEFLUOR 버퍼의 2.8 ML에 튜브 # 7 펠렛을 Resuspend. 항상 얼음에 튜브 보관하십시오. </li><li> 스트레이너 모자 12 X 75mm의 폴리스티렌 테스트 튜브를 사용하여 35 μm의 필터를 통해 세포를 전달합니다. </li></ol> 5. 형광 활성 세포별로 정렬 암 줄기 세포를 분리 <ol><li> FACSAria 흐름 cytometer은 세​​포 분류를위한 준비가되어 있습니다. </li><li> 보정 컨트롤 튜브 # 1, 2, 3, 4, 5의 세포를 사용하여 설정됩니다. </li><li> 게이츠는 셀 singlets를 수집 전달 및 사이드 산란 매개 변수를 기반으로 만들어집니다. </li><li> 비 마우스 (PerCP - 음수)과 가능한 (비 propidium 요오드화물 얼룩) 세포는 FL - 3 채널을 사용하여 문이 있습니다. </li><li> DEAB - 대우 전지 (튜브 # 6) 및 FL - 1 채널을 사용하여 만든 ALDH + 세포는 게이츠에 따라 수집하고 있습니다. </li><li> CD44 + CD24 + 세포는 게이츠에 따라 수집하는 FL - 4 및 FL - 2 채널을 사용 ALDEFLUOR, IgG 2bΚ - APC 및 IgG 2aκ - PE (튜브 # 6) 물들일 세포를 사용하여 만들었습니다. </li><li> 세포 10% FCS와 보충 DMEM 1 ML을 포함 12 X 75mm의 폴리스티렌 테스트 튜브에 수집하고 있습니다. </li><li> 세포가 정렬되고 나면, 가만히 따르다 10 분 미디어 450x g에 세포 현탁액을 원심 분리기, 500 μL 신선한 DMEM에있는 세포를 resuspend 10% FCS와 함께 보충. </li><li> 로 단계 3.8에 설명된 hemocytometer를 사용하여 정렬 세포의 수를 계산합니다. </li></ol> 6. 대표 결과 이 프로토콜은 ALDH + 및 CD44 + CD24 + 췌장 CSCs의 고립으로 이어질 것입니다. 마우스 파생 세포의 비율이 변수이지만, 우리는 대부분의 인간의 췌장 암 xenografts가 마우스 CD31을 사용하여 마우스 유래 세포, 마우스 가계 칵테일, 마우스 H - 2Kd 비오틴 - 복합 항체 (그림 1A 20-60 %를 포함하는 것으로 나타났습니다 ). 마찬가지로 다른 xenografts에서 각 CSC 인구의 빈도가 변수이지만, 일반적으로 우리는 전체 세포 인구 1-4 %가 ALDH + (그림 1B)이며 0.2-5 %가 CD44 + CD24 + (그림 1C) 것으로 나타났습니다. FACSAria 기계 카운트가 자주 인해 FACSAria 감지되지 않은 세포 입자에 정확 있기 때문에 우리는 정기적으로 수동으로 hemocytometer를 사용하여 정렬 셀을 계산합니다. <img src="/files/ftp_upload/2169/2169fig1.jpg" alt="그림 1" /> 그림 1. 유동세포계측법의 줄거리는 췌장암의 이종 이식에서 특정 인구를 묘사. (A) FL - 3 채널에 적극적으로 얼룩 세포 (propidium 요오드화물은 +, 마우스 CD31 + 마우스 가계 +, 또는 마우스 H - 2Kd +)가 단 분리만큼 제외됩니다 가능한이 아닌 마우스 세포를 파생. (B) 인간의 췌장암의 이종 이식의 ALDH 활동은 ALDH1 억제제 diethylamino - benzaldehyde (DEAB)의 존재와 부재의 ALDEFLUOR 시약을 사용하여 유동세포계측법에 의해 측정되었다. 프레임 DEAB로 치료 세포를 기반으로 만든 후 세포 치료에 적용되었다 ALDH + 세포를 묘사 문을 나타냅니다. ALDH의 + 세포의 비율은 게이트에 표시됩니다. (C) CD44 + CD24 + 문이 ALDEFLUOR 물들일 세포를 기반으로 작성하고, IgG 2bκ - APC (CD44 - APC에 대한 isotypic 제어)과 IgG 2aκ - PE는 (CD24 - PE에 대한 isotypic 제어) 항체되었습니다. CD44 + CD24 + 세포의 비율은 게이트에 표시됩니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

이 프로토콜은 인간 xenografts에서 췌장 CSCs의 고립을 설명합니다. 이 절차의 몇 가지 주요 단계는 가능한 CSCs의 수율을 향상시킬 것입니다. 췌장 CSCs의 순수한 인구의 복구 FACSAria에 정렬하기 전에 세포 현탁액에있는 가능한 세포의 순도에 따라 달라집니다. 최대 지름 1 cm 미만 xenografts를 사용 돌보는 것은 종양의 중심부에 괴사 조직의 양을 줄이는 데 도움이됩니다. 또한 Ficoll - Paque 기울기 원심 분리하는 동안 조직 파편과 죽은 세포의 세포 현탁액을 파괴하는 것은 중요하고 세포가 hemocytometer를 계산하는 경우 조직 파편이나 비 가능한 세포의 많은 수의가 감지하는 경우 반복 수 있습니다. 얼룩 프로토콜 여기에 특정 세포 표면 항원을 감지하는 ALDH 활동뿐만 아니라 형광단 - 복합 항체를 감지 ALDEFLUOR 시약 시스템을 활용하여 설명했다. ALDEFLUOR의 얼룩은 37 ° C 수행하고, 따라서 항체 얼룩이 (얼음) 37에서 일어날 수있는 항체가 상한의 가능성, ° C.을 방지하기 전에 완료해야합니다 세포 ALDEFLUOR 시약을 유출 수 있기 때문에 4 ALDEFLUOR 버퍼에있는 세포를 유지하는 것이 중요합니다 ° ALDEFLUOR의 얼룩 후 C가 완료되었습니다. ALDEFLUOR 버퍼는 ALDEFLUOR의 세포내 수준을 유지하는 데 도움이 약물 유출 억제제가 포함되어 있습니다. 이 방법은 가능한 췌장 CSCs를 분리 이후 그들은 이러한 세포의 생리 기능을 측정하는 실험의 숫자 적용할 수 있습니다. 우리는 immunocompromised 쥐 및 체외 세포 마이 그 레이션 / 침공 assays를 이용한 종양 - 개시 assays 이러한 세포를 사용했습니다. 또한, RNA, DNA, 그리고 단백질은 CSC 및 비 - CSC의 인구를 비교 분석 연구에 대해 이러한 세포에서 수집한 수 있습니다.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>DMEM </td>
<td>&nbsp;</td>
<td>Invitrogen (Carlsbad, CA) </td>
<td>11965-118 </td>
<td>&nbsp;</td>
<tr>
<td>Fetal calf serum </td>
<td>&nbsp;</td>
<td>Harlan (Indianapolis, IN)</td>
<td>BT-9501</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-streptomycin </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15140-122 </td>
<td>&nbsp;</td>
<tr>
<td>Collagenase type IV </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>17104-019 </td>
<td>&nbsp;</td>
<tr>
<td>Dispase </td>
<td>&nbsp;</td>
<td>Sigma (St. Louis, MO) </td>
<td>D4818 </td>
<td>&nbsp;</td>
<tr>
<td>100 x 20 mm culture dish </td>
<td>&nbsp;</td>
<td>BD Biosciences (San Jose, CA) </td>
<td>353003 </td>
<td>&nbsp;</td>
<tr>
<td>70-&mu;m filter </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352350 </td>
<td>&nbsp;</td>
<tr>
<td>50-mL conical tube </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352070 </td>
<td>&nbsp;</td>
<tr>
<td>Ficoll-Paque Plus </td>
<td>&nbsp;</td>
<td>GE Healthcare (Upsala, Sweden) </td>
<td>17-1440 </td>
<td>&nbsp;</td>
<tr>
<td>ALDEFLUOR reagent system </td>
<td>&nbsp;</td>
<td>Stem Cell Technologies (Vancouver, BC, Canada) </td>
<td>01700 </td>
<td>&nbsp;</td>
<tr>
<td>Trypan blue </td>
<td>&nbsp;</td>
<td>Invitrogen </td>
<td>15250-061 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352054 </td>
<td>&nbsp;</td>
<tr>
<td>CD44-APC (clone G44-26) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>559942 </td>
<td>&nbsp;</td>
<tr>
<td>CD24-PE (clone ML5) </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555428 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse CD31-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553371 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse lineage-biotin </td>
<td>&nbsp;</td>
<td>Miltenyi Biotec (Auburn, CA) </td>
<td>130-092-613 </td>
<td>&nbsp;</td>
<tr>
<td>Mouse H-2Kd-biotin </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>553564 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2b&#x03BA;-APC</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555745 </td>
<td>&nbsp;</td>
<tr>
<td>IgG2a&#x03BA;-PE</td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>555574 </td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin-PerCP protein </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>554064 </td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide </td>
<td>&nbsp;</td>
<td>Sigma </td>
<td>P4170 </td>
<td>&nbsp;</td>
<tr>
<td>12 x 75 mm polystyrene test tubes with strainer caps </td>
<td>&nbsp;</td>
<td>BD Biosciences </td>
<td>352235 </td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of stem cells from human pancreatic cancer xenografts

암 줄기 세포 (CSCs)이 malignancies의 증가에 확인된하고 기능 자기 갱신을 받아야하고 차별화된 자손 하나를 생산하는 능력에 의해 정의됩니다. 이러한 속성은 immunocompromised 생쥐에 주입했을 때 CSCs 원래 종양 요점을 되풀이 수 있습니다. 상피 강한 악의 이내 CSCs 먼저 유방암에 설명된 및 특정 세포 표면 항원 발현 (CD44 + CD24 낮은 / -) 표시 발견된 2. 이후 CSCs는 CD44과 CD24뿐만 아니라 다른 표면 항원의 번호를 사용하여 다른 사람 malignancies의 증가에서 확인되었습니다. 알데히드 탈수소 효소 (ALDH) 활동을 포함한 Physiologic 속성, 또한 3-5 악성 조직에서 CSCs을 분리하는 데 사용되었습니다. 최근 우리와 다른 ALDH 활동과 세포 표면 항원 CD44와 CD24, CD133과 6-8의 표현에 따라 췌장 선암에서 CSCs을 확인. 이러한 고도로 tumorigenic 인구하거나 수 중복 및 기타 기능이 표시되지 않을 수 있습니다. 우리는 발견 ALDH + 및 CD44 + CD24 + 췌장 CSCs가 비슷하게 tumorigenic하지만, ALDH + 세포가 상대적으로 더 많은 침략 8. 이 프로토콜에서는 낮은 통로 인간 xenografts 9 시부터 가능한 췌장 CSCs을 분리하는 방법을 설명합니다. Xenografted 종양은 생쥐에서 수확 및 단일 세포 현탁액으로 만들어집니다. 조직 파편과 죽은 세포는 살아 세포에서 분리 후 CD44과 CD24에 대한 항체를 사용하고 ALDEFLUOR 시약, ALDH 10 형광 기판을 사용하여 얼룩 있습니다. CSCs 그때 형광 활성 세포 분류에 의해 격리됩니다. 절연 CSCs 그런 다음 가능한 세포를 필요로하는 분석이나 기능 assays 사용할 수 있습니다.

Watch this Scientific Journal Video about Isolation of Stem Cells from Human Pancreatic Cancer Xenografts at JoVE.com

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

암 줄기 세포 (CSCs)이 malignancies의 숫자에서 확인되었습니다. 이 프로토콜에서 우리는 인간의 췌장 선암의 xenografts에서 CSCs을 분리하는 알데히드 탈수소 효소의 활동과 CD44과 CD24 표현을 활용 흐름 cytometric 방법을 설명합니다. 이 가능한 전지는 다음 기능 분석 연구에 사용할 수 있습니다.

인간 췌장암의 Xenografts에서 줄기 세포의 분리

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

isolation-of-stem-cells-from-human-pancreatic-cancer-xenografts

Research

JoVE Journal

Biology

22.2K 조회수.  Johns Hopkins University School of Medicine. 암 줄기 세포 (CSCs)이 malignancies의 숫자에서 확인되었습니다. 이 프로토콜에서 우리는 인간의 췌장 선암의 xenografts에서 CSCs을 분리하는 알데히드 탈수소 효소의 활동과 CD44과 CD24 표현을 활용 흐름 cytometric 방법을 설명합니다. 이 가능한 전지는 다음 기능 분석 연구에 사용할 수 있습니다.

비디오: Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

태반에서 조혈 줄기 세포의 분리 및 분석

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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생물학

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

인간 배아 줄기 세포에서 염색질의 Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

인간 췌장 아일릿 격리 : 부품 I : 소화 및 췌장 조직의 수집

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

사람의 피부에서 성인 상피 줄기 세포의 분리 및 문화

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

인간 유도 Pluripotent 줄기 세포의 선택 및 분리 식민지는 성인 섬유아 세포에서 다시 프로그램

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

일차 종양에서 면역 세포의 분리

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

인간의 기본 섬유 아세포 배양에서 순진 성인 줄기 세포 분리

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

인간 Lipoaspirates에서 지방 유래 줄기 세포를 수동으로 분리

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

성인 마우스 앞니의 치아 상피 줄기 세포의 분리 및 문화

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

인간 혈청에서 작은 비 암호화 RNA를의 분리

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...