우리는이 작업에 참여할 의지의 마이애미 WIHS 연구 참가자 감사합니다. 이 논문의 데이터는 마이애미 여성 부처 HIV 연구 (WIHS)에 의해 수집 하였다. 이 책의 내용은 전적으로 저자의 책임이며 국립 보건원 (NIH)의 공식 견해를 대변하지 않습니다. 이 작품은 전진에 대한 알레르기 및 전염병 국립 연구소와 여자의 부처 간 HIV 감염 연구 (WIHS) 허가 번호 U01의 AI103397, 알레르기 국립 연구소 감염증, 건강 [허가 번호 P30AI073961]의 국립 연구소, 국립 센터 지원 건강의 병진 과학 및 소수 민족 보건 및 건강 불균형에 국립 연구소, 건강 [허가 번호 UL1TR000460 및 1KL2TR000461] 국립 연구소 및 아동 건강 및 인간 발달의 국립 연구소 국립 연구소 [허가 번호 K23HD074489].

연구 활동은 마이애미 여성의 HIV 부처 연구 (WIHS) 및 AIDS 연구를위한 마이애미 센터 (CFAR) .Institutional 검토위원회 (의과 대학 마이애미 밀러 학교) 승인 전에 얻었다와 공동으로 마이애미 HIV 연구 단위의 대학에서 일어났다 채용 및 평가 또는 연구 관련 절차. 1. 자궁 경부 브러쉬 샘플 컬렉션 참고 : 참가자는 훈련 MD 또는 산부인과 의사와 cytobrush를 삽입하여 검경 시험에서 수행되었다 상피내 림프구의 컬렉션에 의해 수행되는 질 검사를 시행 하였다. <ol><li> 참가자 특성 <ol><li> 성적, 활성 임신하지 아니라 피임 약물이나 자궁 내 장치와 나이, 18 ~ 45 년 세되는 여성 참가자를 모집. </li><li> 질 검사하기 전에,이 참가자는 녹색에서 10 mL의 혈액 무승부를 받다말초 혈 단핵 세포 (PBMC) 분리 용 상부 헤파린 vacutainers. 자궁 경부 상피내 분석 (9)에 대한 제어 역할을합니다. </li></ol></li><li> 자궁 경부 샘플 컬렉션 <ol><li> 윤활없이 질에 적절한 크기의 살균 검경를 삽입합니다. 검기와 점액을 청소하는 멸균 거즈의 삽입을 용이하게하기 위해 따뜻한 물을 사용합니다. 검기의 위치는 운영 체제 및 ectocervix의 완벽한 시각화 할 수 있습니다. </li><li> 손에 가장 가까운 단지 강모가 표시 될 때까지 자궁 경관에 자궁 경부 브러쉬를 삽입합니다. 360 °의 브러시를 시계 방향으로 돌립니다. IMDM 배지 5 ㎖를 함유하는 15 ㎖의 튜브에 브러쉬를 옮긴다. </li><li> 즉시 수집 한 후, 얼음에 cytobrush를 포함하는 튜브를 배치합니다. 수집 된 상피내 세포의 높은 생존 능력을 얻으려면 1 시간 내에 실험실 및 프로세스에 cytobrush을 제공합니다. </li></ol></li></ol><ol><li> 4 단 (10 초) 스트로크를 적용 경부 cytobrush을 포함 소용돌이 관. </li><li> X 250 g에서 10 분 동안, cytobrush을 포함하는 튜브를 원심 분리기. </li><li> 조심스럽게 튜브에서 cytobrush을 제거 (튜브 하단의 펠렛을 방해하지 않음) 및 IMDM 미디어의 1 mL를 넣고 얼음에 튜브를 배치합니다. </li><li> 50 ML 튜브의 상단에 100mm의 셀 스트레이너에 cytobrush를 놓습니다. </li><li> 20 ㎖의 IMDM으로 cytobrush을 씻으십시오. </li><li> 250 × g에서 10 분 동안 원심 분리기 튜브. </li><li> 뜨는을 가만히 따르다 및 펠렛 세포를 1 mL의 IMDM에 재현 탁 2.3에 설명 된 이미 다시 중단 펠렛과 결합. </li><li> 자동 세포 계수기 (10)를 사용하여 세포를 트립 판 블루 수나 염료 10 μL로 세포 현탁액 10 μL를 조합하여 혈구를 이용하여 세포 수를 계산. 부드럽게 피펫 및 열 배 아래로 세포를 혼합 염색. 로드 10 μL두 혈구 챔버들 중 하나의 개구 내로의 혼합물. 다섯 개의 격자 영역의 모든 세포를 카운트. 생존과 비 생존 세포의 별도의 수를 유지합니다. 다음 식을 사용하여 세포 생존도를 결정 생존 세포 % = 비 생존 세포 × 100 / 총 세포 수의 세포를 포함한다. 다음 계산을 사용 mL의 후속 세포 농도 (세포의 총 수)를 결정한다. ml의 당 전체 세포는 = 총 세포 계수 × (희석 배수 / 계산 사각형의 수) 10,000 세포 / ml를 X. </li><li> 10 % DMSO 90 % FBS를 포함하는 동결 매체를 사용하여 격리 된 자궁 경부 세포를 고정. </li></ol> 3. 유동 세포 계측법 <ol><li> PBS를 1 % 소 혈청 알부민을 함유하는 100 μL FACS 완충액에 1-3 × 105 자궁 경부 세포 (다시 일시 </li><li> 1 μL / 10 추가 6 LIVE / DEAD 고칠 옐로우 사균 얼룩 키트 및 표 1에 기재된 농도로 표면 마커에 대한 항체. </li><li> 빛으로부터 보호 4 ° C에서 30 분 동안 세포를 품어. </li><li> 350 XG에서 5 분 동안 1 ㎖의 FACS 완충액 및 세포를 원심 분리 관에 추가 </li><li> 300 μL 1 % 파라 포름 알데히드의 세포 펠렛을 다시 일시 중지합니다. </li><li> FACS 완충액 100 μL 비드 한 방울 샘플 염색에 사용 된 항체의 동일한 농도를 첨가하여 염색에 사용되는 비드 및 항체를 사용하여 보정 제어를 준비한다. </li><li> 사이토, 405 nm의, 488 nm의 및 635 nm의 레이저가 장착 된 흐름에 스테인드 구슬과 샘플을 획득. </li></ol> 4. 자궁 경부 감마 델타 T 세포 분리 <ol><li> 자기 비드 분리 <ol><li> 다시 중단 재료 및 시약의 표 참조 TCR 감마 델타 마이크로 비드 키트에 포함 된 버퍼의 40 μL에서 2.6에 설명 된 자궁 경부 세포 펠렛 (≤10 7 총 셀). </li><li> 두 단계 염색 프로토콜에 대한 키트의 지시 사항을 따르십시오 : 첫째, 재치항 합텐 마이크로 비드-FITC와 시간 반 TCR 감마 델타 합텐 - 항체 및 초, </li><li> 4-8 ℃에서 15 분간 배양 한 후, 10 분 동안 300 XG에 버퍼 원심 1 ㎖를 첨가하여 세포를 세척 하였다. 튜브를 반전시켜 상등액을 경사 분리하고, 종이의 도움으로 건조 탭. </li><li> 제공된 버퍼 500 μL의 세포 펠렛을 다시 중단하고 공급 업체의 프로토콜에 따라 적절한 자기 분리기에 배치하여 자기 열을 준비합니다. </li><li> 칼럼에 세포 현탁액을 적용하고 통과 레이블이없는 세포를 수집하고 버퍼의 적절한 금액 (500 μL)과 열 배 씻는다. 버퍼 세 번, 열 저수지가 비어 번 할 때마다 추가하여 세척 단계를 수행합니다. 총 폐수를 수집합니다. 이 레이블이없는, 음의 세포 분획이다 </li><li> 열이 컬럼에 버퍼 1 ㎖를 자기 분리기 및 피펫을 형성하고 즉시 자기 라벨 부분을 세척 제거단단히 열와 함께 제공된 플런저를 적용하여 세포를 에드. 주의 : 자성 표지 항 합텐 마이크로 비드는 형광 염료 FITC가 결합되어 있기 때문에 절연 감마 델타 T 세포는 이미 형광 유세포 분석을 위해 염색 하였다. </li></ol></li><li> 셀 정렬 <ol><li> 생존 다이 항체 패널 셀 정렬, 라벨 자궁 경부 세포의 경우 3 절에 설명 된대로. </li><li> 라이브, CD45 + CD3 + 감마 델타 양성 세포에 전자 게이트를 설정하고 셀 정렬을 수행합니다. </li></ol></li></ol>

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저자는 이해의 충돌을 야기 할 수 상업적 또는 다른 관련이없는 (예를 들어, 제약 사주, 컨설팅, 자문위원회 회원, 관련 특허 또는 연구 자금).

여성 하부 생식기의 점막 취약점 평가는 HIV 수집 및 전송의 위험을 다루는 임상 연구의 필수적인 구성 요소입니다. 일반적으로 HIV 감염에 FGT 취약점은 생식기 분비물 (사이토 카인, 케모카인 및 항균 펩타이드) 12, 13에 용해 면역 조절제를 측정하여 평가한다. 그러나 이러한 바이오 마커는, 질 염증의 지표이다 생리 학적 및 병리학 적 사건에 의해 영향?...

우리는 GD 상피내 T 세포를 평가하기 위해 브러시 경부 샘플을 사용하는 방법을 개발 하였다. 자궁 경관은 원주 상피의 단일 층으로 늘어서있다. 상피내 림프구는 병원성 미생물 7, 8 발생에 면역 반응을 빠르게을 시작할 수 있습니다 상피 층 내부에있는 전선 림프구를 나타냅니다. 자궁 경부 상피내 구획의 면역 학적 사건을 이해하는 것은 HIV를 포함하여 감염을 방지하는 효과적인 전략의 설계에 중요합니다. 우리의 방법은 HIV 감염 또는 HIV 감염 위험이있는 여성 GD 상피내 T 세포의 역할을 조사하기 위해 신선하게 수집 된 인간 경부 샘플뿐만 아니라 냉동 경부 세포에 적용 할 수있다. 이 프로토콜의 주요 목표는 자궁 경부 상피내 구획에 새로운 면역 학적 이벤트를 발견하고 evaluat하는 것입니다HIV 감염 여성의 점막 취약점 마커로 전자 GD T 세포 반응. FGT 내성 주변 지식에 상당한 격차가 성공적으로 수집하고 점막 샘플의 처리에 어려움에 부분적으로 기인한다. 또한, 복잡한 점막 면역 세포 이질성, 서로와의 상호 연결성은 면역 시스템의 상태 및 성능을 반영 임상 적 측정을 식별하는 주요 과제이다. 우리는 추가 될 FRT 면역 기전을 확인하기위한 없어서는 안될 매우 다중화 된 단일 셀 기술을 사용하여 분석 할 수 고순도 GD 상피내 T 세포를 수득하는 방법을 개발 하였다.

<table border="0" cellpadding="0" cellspacing="0" width="648">
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			<td height="42" style="height:42px;width:216px;">Cytobrush Plus GT&#160;</td>
			<td style="width:147px;">(CareFusion, San Diego, CA, USA</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="42">
			<td height="42" style="height:42px;">IMDM,Iscove&#39;s Modified Dulbecco&#39;s Medium&#160;</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">12440061</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Beckman Coulter automated cell counter (The Vi-CELL Series Cell Viability Analyzer</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td align="right" style="width:119px;">496178</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO, Dimethyl sulfoxide&#160;</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS, Fetal Bovine Serum</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td style="width:119px;">26140-079</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:147px;">Invitrogen, Gibco</td>
			<td align="right" style="width:119px;">10010023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BSA</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A-9647</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">LIVE/DEAD Fixable Yellow Dead Cell Stain kit&#160;</td>
			<td style="width:147px;">Life Technologies,</td>
			<td style="width:119px;">L349S9</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1% paraformaldehyde (Sigma-Aldrich</td>
			<td style="width:147px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">D6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fortessa flow cytometer&#160;</td>
			<td style="width:147px;">Becton Dickinson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraComp eBeads</td>
			<td style="width:147px;">eBioscience</td>
			<td style="width:119px;">01-2222-42</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">ArC Amine Reactive Compensation Beads (Life Technologies, Grand Island, NY, USA)</td>
			<td style="width:147px;">Life Technologies</td>
			<td style="width:119px;">A10346</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-TCR gamma/delta MicroBead Kit&#160;</td>
			<td style="width:147px;">Milteny Miltenyi Biotec Inc.</td>
			<td style="width:119px;">130-050-701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS column</td>
			<td style="width:147px;"></td>
			<td style="width:119px;">103-042-201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MACS separator</td>
			<td style="width:147px;"></td>
			<td align="right" style="width:119px;">4124</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric analysis of human endocervical gamma delta t cells

여성의 생식 기관 (FRT) 점막 면역 시스템은 첫 번째 방어선 역할을합니다. 성기 점막의 더 나은 지식은 HIV를 포함하여 다른 병원균의 병원성을 이해하는 것이 필수적이다. 감마 델타 (GD) T 세포는 &#39;비 전통적인&#39;T 세포의 원형이며, 감마 및 델타 사슬로 구성된 이종이 T 세포 수용체 (TCR도)들의 식으로 정의 T 세포의 상대적으로 작은 부분 집합을 나타낸다. 이 알파 - 베타 TCR도에 의해 정의 된 고전 훨씬 더 잘 알려 CD4 + 헬퍼 T 세포와 CD8 + 세포 독성 T 세포에서 차별화. GD T 세포는 종종 조직 별 현지화를 보여 상피 세포에 농축되어있다. GD T 세포는 염증, 종양 감시, 감염성 질환, 및자가 면역 반응에 대한 면역 반응을 조율. 여기서는 재현성 분리 및 인간 자궁 경부 상피내 GD T 림프구를 분석하는 방법을 제시한다. 우리는 하여자의 부처 간 HIV 감염 연구 (WIHS)에 참여하는 여성에서 사용 경부 cytobrush 샘플을했습니다. 질 점막 모독가 인 조건에서 GD T 세포의 상호 작용에 대한 지식은 질 microbicides.In 추가의 개발을 포함한 점막 취약점을 해결하는 임상 연구에 적용 할 수 점막 GD T 세포 반응에 대한 지식을 가지고 감염성 질환의 치료에서 GD T 세포 기반 면역 요법의 적용 가능성.

최근에는 경부 상피내 GD T 세포를 분석하고, 우리는 HIV 감염 여성 9, 11의 경부 감마 델타 T 세포의 감소에 관하여보고 처음이었다. 여기서는 분리하고 경부 감마 델타 T 세포의 서브 세트를 분석하는 프로토콜을 기술한다. 도 1에 도시 된 바와 같이, T 세포 GD 용이 인간 자궁 경부 세포 샘플에서 검출 될 수있다. <p c...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

인간의 자궁 경부 감마 델타 T 세포의 분리 및 유세포 분석

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

isolation-flow-cytometric-analysis-human-endocervical-gamma-delta-t

Research

JoVE Journal

Immunology and Infection

8.7K 조회수.  Miller School of Medicine, University of Miami. We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

비디오: Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular matrix proteins. The precise spatiotemporal activation of integrins from a low affinity state to a high affinity state at the cell leading edge is important for T lymphocyte migration 1. Likewise, retraction of the cell trailing edge, or uropod, is a necessary step in maintaining persistent integrin-dependent T lymphocyte motility 2. Many therapeutic approaches to autoimmune or inflammatory diseases target integrins as a means to inhibit the excessive recruitment and migration of leukocytes 3. To study the molecular events that regulate human T lymphocyte migration, we have utilized an in vitro system to analyze cell migration on a two-dimensional substrate that mimics the environment that a T lymphocyte encounters during recruitment from the vasculature. T lymphocytes are first isolated from human donors and are then stimulated and cultured for seven to ten days. During the assay, T lymphocytes are allowed to adhere and migrate on a substrate coated with intercellular adhesion molecule-1 (ICAM-1), a ligand for integrin LFA-1, and stromal cell-derived factor-1 (SDF-1). Our data show that T lymphocytes exhibit a migratory velocity of ~15 &mu;m/min. T lymphocyte migration can be inhibited by integrin blockade 1 or by inhibitors of the cellular actomyosin machinery that regulates cell migration 2.

Human T Lymphocyte Isolation, Culture and Analysis of Migration In Vitro

T lymphocyte migration occurs during homing to lymphoid organs, exit from the vasculature, and entering into peripheral tissues. Here, we describe a protocol that can be used to analyze T lymphocyte migration in vitro.

마이 그 레이션의 인간 T 림프구 분리, 문화 및 분석 체외에서</em

The migration of T lymphocytes involves the adhesive interaction of cell surface integrins with ligands expressed on other cells or with extracellular ...

연구

면역학 및 감염병학

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

기능 및 분자 연구 기본 손쥐 유형 II 폐포 상피 세포의 흐름 Cytometric 절연

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

나노 입자 백신 접종 후 분석 항원 특이 CD8 + T 세포 반응에 대한 전체 동물 이미징 및 유세포 기술

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

허혈성 마우스 뇌에서 면역 세포의 분리 및 유세포 분석

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

분리 및 신경 교종-침투 말초 혈액 단핵 세포의 유세포 분석

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

쥐 소장 림프구의 분리 및 유세포 특성

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

PM10의 입자 바인딩 베팅 1 절 알레르기의 유세포 분석

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

인간의 전체 혈액 자연 살해 세포 용 균성 활동의 유세포 분석

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

고립과 Murine 인간과 치경 대 식 세포의 생체 외에서 문화

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

림프 내 피 세포의 흐름 Cytometric 분석 Murine 간 소화

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...