The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

윤리 문 : 매니토바 대학교 및 나이로비의 케냐 타 국립 병원 / 대학 모두의 연구 윤리 보드는이 연구를 승인하고 서면 동의서는 모든 연구 참가자로부터 얻은 것입니다. 1. 매체의 제조 및 CMC 수집 관 <ol><li> 인산염 완충 생리 식염수 (PBS) 용액 (137.93 mM의 염화나트륨, 2.67 밀리미터의 KCl, 8.1 mM의 나 2 HPO 4, 1.47 mM의 KH 2 PO 4)를 준비합니다. 불임에 대한 압력솥. 이것은 몇 달 동안 4 º C에 저장할 수 있습니다. </li><li> 미리 나누어지는 5 ㎖의 PBS로 50 ML 팔콘 튜브 (수집 할 샘플 당 하나)와 4 º C에서 저장 샘플 수집 시간까지. </li><li> 저장을위한 세척을 수집하는 샘플 당 2 ~ 4 cryovials 또는 1.5 ML의 microcentrifuge 튜브 레이블을 지정합니다. </li><li> 세포 배양 매체를 준비 RPMI 1640 + 1 % 페니실린 / 스트렙토 마이신 / 암포 테리 신 B (최종 농도 100 단위 / ㎖, 100 ㎍ / ㎖ 및 250 겨 / ㎖, respectiv더 FCS / FBS와 엘리는 덧붙였다. </li></ol> Cytobrush 샘플 2. 컬렉션 cytobrush 샘플 컬렉션은 훈련 MD 또는 산부인과 의사에 의해 수행되어야하는 비 침습 절차입니다. <ol><li> cytobrush와 나무 또는 플라스틱 긁는 도구를 모두 사용하여 자궁 경부 단핵 세포 (CMC)를 수집합니다. (그림 1) 긁는 도구를 삽입하고 자궁 OS 중심으로 회전하여 검경 시험에서 참가자에서 CMC를 수집합니다. , 자궁 경부 OS에 cytobrush를 삽입 360 ° 회전, 즉시 PBS의 5 ML을 포함하는 50 ML 팔콘 튜브에 cytobrush 스크레이퍼를 모두 배치합니다. </li><li> 처리까지 4 º C에서 / 얼음에 샘플을 보관하십시오. 컬렉션의 2 시간에서 세포 생존 능력, 공정 샘플을 유지합니다. </li><li> 가능하면 말초 혈액 단핵 세포 CMC 분석을위한 비교 / 제어 역할을하는 (PBMC) 분리를위한 녹색 최고 헤파린 vacutainers에 일치하는 혈액 샘플을 수집합니다. </리&gt; </ol> CMC를 3. 분리 이중 장갑 멸균 생물 안전 캐비닛, 바이오 안전성 수준이 실험실에서 시료 처리를 수행합니다. <ol><li> 수집하는 동안, CMC 샘플은 혈액에 오염 될 수 있습니다. 최종 샘플 PBMC의 혼란 포함을 방지하기 위하여 연구 결과에서 볼 혈액 오염과 샘플을 제외. 인해 CMC 샘플로부터 단리 림프구의 낮은 숫자로, 사소한 혈액 오염을 쉽게 CMC 림프구 성분을 압도 할 수있다. </li><li> 브러쉬에서 세포를 떼어 놓다 45 초 cytobrush 스크레이퍼를 모두 포함 소용돌이 팔콘 튜브. 샘플은이 과정에서 거품이 될 수있다; 이것은 정상입니다. </li><li> 스크레이퍼 떨어져 남아있는 재료를 긁어 및 표백 용액에 스크레이퍼를 폐기 cytobrush를 사용합니다. 브러쉬 / 스크레이퍼에 붙어 남아있는 세포를 수집하려면, 엄지 손가락과 집게 손가락 사이에 스크레이퍼를 짜내 신선한 장갑을 사용합니다.세포와 PBS가 동일한 50 ㎖ 튜브에 수집 될 수 있도록 브러시를 밀어. 표백제 용액에 cytobrush를 폐기하고, 장갑을 버린다. 중요 : 각 샘플에 대한 새로운 장갑을 사용하십시오. </li><li> 신선한 50 ML 팔콘 튜브에 100 μm의 나일론 셀 스트레이너를 장착한다. 전달 피펫을 사용하여, 샘플 튜브에서 PBS-세포 현탁액을 수집하고 새로운 튜브로 여과기를 통해 고를. </li><li> 원래 샘플 튜브에 RPMI 1640 5 ML을 추가합니다. 전송 피펫을 사용하여 RPMI로 샘플 튜브의 측면을 씻어 새 컬렉션 튜브에 필터를 통해 전송합니다. 뿐만 아니라 RPMI와 나일론 필터의 바닥을 세척 할 것. </li><li> 514 X g에서 10 분 동안 원심 분리. 이것은 CMC 펠릿 빠지을 방지하기 위하여,이 단계 동안 원심 브레이크를 남겨두기 위하여 중요하다. </li><li> 조심스럽게 펠렛을 방해하지 않도록주의하면서 튜브에서 뜨는을 제거합니다. 펠렛의 크기는 매우 다양 할 것이다; 그래서의다른 경우에, 펠릿이 거의 보이지 및 고투명 동안 저 샘플은 펠릿 인해 상피 세포의 많은 수의 존재로 상당히 크다. </li><li> 팔콘 튜브의 부드러운 교반하여 펠렛을 Resuspend. 샘플을 씻어 PBS의 5 ML을 추가합니다. </li><li> 아니 원심 브레이크와 514 XG에 10 분 다시 원심 분리기. </li><li> 조심스럽게 뜨는을 취소하고 위의 펠렛을 다시 일시 중지합니다. 세포 (PBMC의 냉동 보존 프로토콜을 사용하여) 냉동 보존, 자극 또는 표현형 유동 세포 계측법 중 하나에 대한 준비가되어 있습니다. </li></ol> 4. CMC 표면의 얼룩 및 유동 세포 계측법 <ol><li> 차단 용액 100 μL 3.10 단계에서 펠렛을 재현 탁하고 96 - 웰 플레이트 또는 FACS 튜브 내로 하나 옮긴다. 차단 솔루션 (Fcγ 수용체를 차단하기 위해) 다음과 같은 조리법은 : 1.8 ㎕의 마우스 IgG의 (최종 농도 0.2 ㎍ / μL), 5 μL의 FBS 93.2 ㎕의 FACS 세척 (PBS 2 % FBS).염색법은 펠릿 크기가 충분히 작은 경우에 96 웰 플레이트에서 수행 될 수 있지만, 펠릿 일상적 큰 경우 FACS 튜브에서 수행 될 필요가있다. 그에 따라 항체를 적정하는 것이 중요합니다. </li><li> 4 ℃에서 얼음 / 10 분의 샘플을 차단 </li><li> 96 - 웰 플레이트에 FACS 워시 (PBS 2 %의 FBS) 100 μL, 또는 FACS 튜브에 500 ㎕ 씩 세포를 씻으십시오. 최저 브레이크와 4 ° C에서 10 분 600 XG에 원심 분리기 (끄거나, 낮은 브레이크 설정을 사용할 수없는 경우가). </li><li> 뜨는을 제거하고 교반하여 세포 펠렛을 다시 일시 중지합니다. 생존의 얼룩 (라이브 죽은 고칠 가능성 염료)를 추가합니다. 제조업체의 지침에 따라 생존의 염료를 준비합니다. 각 실험실 / 사이토 설치에 사용하기 위해 적정해야 할 수 있습니다. 4 ° C에서 어둠 속에서 30 분 알을 품다 </li><li> (96 - 웰 플레이트 / FACS 튜브의 경우) 세포에게 PBS 100 μl/500 μl를 씻으십시오. 저 / 노 브레이크 10 분 600 XG에 원심 분리기. supernatan를 제거T와에 resuspend 세포. </li><li> 100 μL의 총 부피, 표면 마커 항체의 칵테일 세포를 품어. 중요 : 최적화 및 염색을 샘플링하기 전에 패널 세포 계측법 각 흐름을 적정한다. 4 ° C에서 어둠 속에서 30 분 동안 세포를 품어 </li><li> 4.5 단계를 반복합니다. FACS 튜브에 96 - 웰 플레이트, 전사 셀에 염색하고 1 % 파라 포름 알데히드 (PFA)를 포함하는 FACS 세척 750 μL의 최종 부피로 희석 (또는 CytoFix을 소재 1 희석). 유동 세포 계수기에 데이터 수집을 진행합니다. </li></ol> 5. 수집 및 자궁 경부 질 세척의 준비 (CVL) <ol><li> 샘플링을 cytobrush하기 전에, 멸균 PBS의 2 ㎖로 자궁 경부 씻어 후방 원개에서 세척을 흡인하기 위해 주사기를 사용합니다. </li><li> 15 ML 팔콘 튜브에 흡입 된 세척 샘플을 수집하고 처리 할 때까지 4 º C에서 / 얼음 계속. </li><li> 세포 파편을 제거하는 7 분 400 XG에 샘플을 원심 분리기. </li><li> 공동-70 ℃에서 1 ㎖ 또는 500 μL 씩 분주하고, 가게에 뜨는 나누어지는에게 샘플을 llect 분취 량은 ELISA 또는 CVL 단백질의 비드 어레이 분석 해동 만 체류 냉동 / 해동 사이클을 피할 수있다. </li><li> 원하는 경우, 제조자의 프로토콜에 따라 RNA의 발현을 측정하기 위해 나중에 RNA에서 세포 파편의 펠렛을 재현 탁. </li></ol> 6. 데이터 수집 <ol><li> 항체 패널 일치 보상 튜브 세트를 준비한다. 스펙트럼 중복을 조정 보상 튜브를 실행합니다. 그 사이토 어떤 여러 가지 빛깔의 흐름에 샘플을 실행은 패널에 사용되는 형광 색소 결합 된 항체에 대해 구성됩니다. </li><li> 림프구 집단을 검출하기 위해 순방향 캐터 (FSC) 및 측면 스 캐터 (SSC)의 전압을 조정하는 제 PBMC 시료를 획득. 리니어 스케일의 각 축 (100)에 그들을 유지하십시오. PBMC 제어를 실행하면 상당히 쉽게 CMC 림프구 인구를 찾을 수 있습니다. </li><li> CMC 샘플에 대한 FSC 임계 값은 낮은 FSC의 값에 SSC 축을 얼룩에 의한 PBMC 샘플에 상대적으로 증가해야 할 수 있습니다. </li><li> 취득하기 전에 각 튜브를 소용돌이. 수집 된 림프구 게이트 이벤트의 수를 최대화 할 전체 튜브를 획득. 막힘을 방지하기 위해주의 깊게 시스템을 모니터링합니다. </li><li> 그러한 FlowJo 같은 유세포 분석 프로그램으로 데이터를 내보낼. </li></ol> 7. 게이팅 전략 <ol><li> 고 앞으로 측면 산란 (FSC-H)을 선택 / 앞으로 측면 산란 영역 (FSC-A) 중항 인구를 결정합니다. </li><li> 인수의 품질을 제어하기 위해 (예를 FITC로) 형광 마커 대 시간을 선택합니다. 유량의 변화는 데이터에 유물을 소개 할 수 있습니다. 유량의 차이를 보여줍니다 어떤 지역을 제외합니다. </li><li> SSC-A/FSC-A 플롯의 림프구 인구를 확인합니다. 식별을 용이하게하기 위해 PBMC 컨트롤을 사용합니다. CMC 림프구 인구가 PBMC 제어와 같은 명확하지 않을 수 있습니다. &lt;/ 리&gt; <li> SSC-A/viability에 게이트는 살아있는 세포에서 죽은 세포를 제외 염색. 죽은 세포가 아닌 구체적으로 유물을 소개하는 항체를 통합 할 수 있습니다. </li><li> SSC-A/CD3에 게이트는 T 세포 인구를 식별 할 수 있습니다. 이 게이트에서 CD4 +와 CD8 + 인구를 식별합니다. </li></ol>

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The authors have nothing to disclose.

여성의 생식기 (FGT)에서 내성에 대한 지식의 큰 격차를 감안할 때, CMC를의 표현형 분석은 자궁 경부에있는 다수의 림프구 집단에 대한 통찰력의 다양한 배열을 제공 할 수 있습니다. 프로테옴 분석 및 자궁 세척에서 바이러스 부하 측정과 함께, 성병 감염 (STI)의 다른 병원균에 대한 면역은 다양한 인구에서 해부 할 수 있습니다. 기술적 고려 사항 - CMC를가 : 분리 ...

새로운 HIV 감염의 대부분은 전 세계적으로 여성은 2011 년 (UNAIDS 1) 새로운 감염의 47 %를 대표로, 이성애 전송을 통해 발생한다. 여성의 생식기 (FGT), HIV 및 기타 성병 병원체의 주 진입 포털 중 하나를 이해, 감염을 방지하기 위해 효과적인 전략을 찾는 경로에 높은 중요하다. 생식기 점막의 면역 반응은 분명히 독특하고 말초 혈액 2에서 측정 다릅니다. 그러나, FGT에서 면역 역학의 현재의 지식은 기껏해야 제한됩니다. 현재까지, 점막 면역 환경의 연구는 대부분이 감염 다음과 같은 점막 조직에서 초기 이벤트 이후 질병의 진행 3,4에 큰 영향을 미칠 것이 분명되고있다 창자 관련 림프 조직 (GALT)에 초점을 맞추고있다. 생식기 점막에서 샘플을 수집하는 것은 큰 도전을 나타내고 락 적어도 부분적으로 책임이있다FGT의 면역학의 이해 K. FGT이 로케일에서 샘플을 수집하고 분석하기위한 효율적인 방법을 필요로 한 독특한 환경의 맥락에서 호스트와 병원체의 면역 동적으로 퍼즐을 해결. FGT는 두 개의 섹션으로 나누어 져 나팔관, 자궁 내막과 자궁 경부, 그리고 ectocervix을 포함하는 낮은 요로와 질을 포함하는 상부 생식기 (Kaushic 검토를 등 5). 그것은 서로 다른 사이트의 상대적 기여도는 HIV 감염에 무엇 아직 불분명하지만, 그것은 두 사이트 모두 HIV 항목 6에 기여할 수 있다고 믿고 있습니다. T 세포는 대 식세포가 (로드 리 게스 - 가르시아 검토 등 2) 약 10 %를 포함하는 동안, 상단과 하단의 생식 책자에서 백혈구 40 ~ 50 %를 차지한다. T 세포는 질, 경부, 자궁 내막 및 검출 할 수있다. 대 식세포는 더 강하게 locali 있습니다그들은 두 조직에서 검출 될 수 있지만, 자궁 경부보다 자궁 내막 및 자궁 근층 결합 조직에 제드 자형. 마지막으로, plasmacytoid 수지상 세포 (PDCS) 및 랑게르한스 세포는 또한 FGT 조직에서 검출 될 수있다. 표현형 및 면역 인구 및 HIV 감염에 감수성의 비율은 호르몬주기에 따라 중요하게 다를 수 있습니다, 호르몬 피임약, 세균성 질염이나 성적 활동 5,7-9의 사용. 다양한 방법 FGT의 면역 인구와 환경을 연구하기 위해 개발되었다. 자궁 경부 조직 검사, 자궁 및 자궁 경부 cytobrushes lavages (CVL) 10 ~ 12은 가장 일반적으로 문헌에서 사용할 수 있습니다. PBS의 세척에 의한 CVL 컬렉션은 간단한 방법이며, 매우 낮은 세포 수율 면역 조절 성 단백질 만, 결과의 연구를 허용하고, 따라서 FGT (13)의 면역 세포 집단을 공부에 적합하지 않습니다. CVL 샘플은, 반면에, 아르 V이러한 ELISA, 사이토킨 비드 어레이 (14) 또는 질량 분석 등의 방법 (15, 16)를 사용하여 각종 사이토킨, 케 모킨 또는 항균 인자의 발현을 측정함으로써 FGT의 면역 환경을 평가하기위한 유용한 ERY. 면역 세포 주파수, 표현형과 기능의 특성은 자궁 cytobrush 또는 자궁 경부 생검 표본 추출에 의해 자궁 경부 단핵 세포 (CMC)를 수집하여 달성 될 수있다. 자궁 경부 생검의 샘플링은 출혈의 불편과 위험을 증가와 여자 (12)의 면역 상태에 따라 절차에 따라 치료 2~11일 소요 침습적 방법이다. 한편, 자궁 cytobrushes는 수집 세포의 낮은 수율에도 불구하고, FGT로부터 면역 세포를 수집하기 위해 덜 침습적 더 편리한 방법이다. 두 가지 방법은 CD45 + 백혈구의 동일한 수율에 도달 할 수 있지만, 두 개의 연속 자궁 cytobrushes가 포함 된 세포의 동일한 금액을 얻기 위해 필요한 IN 한 생검 13. 그럼에도 불구하고, cytobrush 샘플링은 여전히 14 유동 세포 계측법에 의해 추가로 생체 표현형에 대한 세포의 수용 수 (약 5,000 CD45 + 세포 / cytobrush)를 제공합니다. 자극 세포 유세포 또는 qPCR에 HIV가 특정 면역 반응 (17) 또는 토륨 셀 (18)의 편광을 식별 cytobrush 유래의 CMC를 사용하여 수행되었다로서 또한 기능적인 특성은,이 샘플들에 수행 될 수있다. T 세포 인구의 팽창은 또한 CMC를 19으로 기능적 연구를 용이하게 할 수있다. 그것은 생검 및 cytobrushes는 FGT의 별개의 부분을 샘플링하는 것이 중요합니다. 자궁 cytobrushes은 자궁 경부와 아마 변환 영역의 상피 세포에서 파생 된 세포를 수집, 자궁 OS를 샘플링하면서 생검은 ectocervix (12, 13)의 상피 세포와 기질의 우수한 부분에서 파생됩니다. Cytobrush 샘플 따라서 재를 샘플링조직 검사는 편평 층상 상피 5 줄 지어 영역을 포함하는 동안 기온은 원주 상피 세포의 단일 층으로 구성. 그 결과, 자궁 경부 생검 및 cytobrush 의해 수집 백혈구 집단의 특성은 다르다. cytobrushes는 CD14 + 단핵 세포 / 대 식세포 (13)의 높은 비율의 컬렉션에 발생하는 반면 생검, CD3 + T 세포의 높은 비율을 수집합니다. FGT의 면역학을 공부하는 것은 많은 년간 20 ~ 22에 대한 관심이었다 우리는 cytobrush 파생 CMC를 연구와 전문 지식의 큰 거래를 축적했다. 우리의 연구는 HIV에 감염된 감염되지 않은 및 HIV에 노출 된 혈청 음성 나이로비, 케냐 (HESN) 여성 성 노동자의 연구에 주로 초점을 맞추고있다. HIV는 우선적으로 FGT에 HIV 대상이 될 수있는 활성화 된 세포의 23 숫자가 낮아 HIV 취득에 대한 보호에 기여할 수있는 활성화 된 T 세포에 복제합니다. 이 가설 여러 스투과 일치에스 아직 감염되지 않은 24, 25을 유지하며,이 대기 표현형도 FGT 14에서 관찰되는 높은 HIV에 노출 된 HESN의 성 노동자들 사이 낮은 면역 활성화를 설명했다. 여기, 우리는 처리 방법을 설명하고 생체 유동 세포 계측법에 의해 자궁 경부 cytobrushes에서 파생 된 CMC 샘플에서 T 세포의 활성화를 평가.

<table border="0" cellpadding="0" cellspacing="0" width="768">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Name of Material/ Equipment</td>
			<td style="width:136px;">Company</td>
			<td style="width:133px;">Catalog Number</td>
			<td style="width:205px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">100uM Cell Strainer for 50 ml Falcon tube</td>
			<td style="width:136px;">BD</td>
			<td style="width:133px;">352360</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">RPMI 1640</td>
			<td style="width:136px;">Hyclone</td>
			<td style="width:133px;">SH30027.01</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fetal Bovine serum&#160;</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">16000044</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Fungizone</td>
			<td style="width:136px;">Life technology</td>
			<td style="width:133px;">15290-018</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Penicillin/streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">P4333-20ml</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">50ml Falcon tube</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">14-959-49A</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Blood Bank disposable transfer pipette</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">13-711-6M</td>
			<td>CMC processing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Cytobrush plus</td>
			<td style="width:136px;">Cooper surgical</td>
			<td style="width:133px;">C0121</td>
			<td>CMC sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Disposable cervical scraper</td>
			<td style="width:136px;">Quick medical</td>
			<td style="width:133px;">2183</td>
			<td>CMC sampling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">15 ml Falcon tube&#160;</td>
			<td style="width:136px;">Fisher&#160;</td>
			<td style="width:133px;">14-959-70c</td>
			<td>CVL processsing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">1.5ml tube ependroff</td>
			<td style="width:136px;">Fisher</td>
			<td style="width:133px;">05-402-18</td>
			<td>CVL storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD Fixable Cell Stain Kit</td>
			<td style="width:136px;">Invitrogen</td>
			<td>Various</td>
			<td>Flow cytometry reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fixation Buffer (4% PFA)</td>
			<td style="width:136px;">BD</td>
			<td>554655</td>
			<td>Flow cytometry regeant&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">IgG mouse&#160;</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:133px;">I8765</td>
			<td>Flow cytometry regeant&#160;</td>
		</tr>
	</tbody>
</table>

collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

다 항목 유동 세포 계측법은 이전에 uncharacterized 조직에서 세포 부분 집합의 표현형과 기능을 해부 할 수있는 강력한 도구입니다. CMC의 샘플 분석은 적절한 게이팅 전략 림프구 및 단핵구 집단 모두에 대한 정보를 얻을 수있다. 대표적인 CMC 게이팅 전략, 일치 PBMC 프로파일에 비해, 그림 2와 같다. FSC-H 플롯 대 FSC-A도 여과 한 후, PBMC에 비해 CMC 샘플에서 매우 널리 ?...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

컬렉션, 격리, 인간 자궁 경부 샘플의 유세포 분석

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.0K 조회수.  University of Manitoba. The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays. 

비디오: Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

부분 Pancreatectomized 환자에서 인간 아일 레츠의 분리

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

연구

의학

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

인간의 췌장을위한 컬렉션 프로토콜

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

인간의 태아 췌장 세포 클러스터의 분리, 문화, 및 이미징

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

신선한 수술 샘플에서 분리 및 인간의 심실 심근 기능 특성

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

지역 사회 기반의 설정에서 획득 샘플의 신속한 분별 및 전체 혈액 성분의 분리

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

격리 및 프로파일 링 마이크로 RNA-포함하는 인간의 담즙에서 엑소 좀을

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

다른 균질화 방법을 통해 마우스 뇌 / 척수에서 검색 된 여러 세포 유형의 동시 유량 세포 측정 특성 분석

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

인간의 혈류를 모방하는 흐름 루프 모델에서 혈액 접촉 임플란트의 혈적합성 테스트

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

작은 모발 샘플에서 11가지 약물 내성 결핵 약물 정량화를 위한 LC-MS/MS 패널 검증

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...