저자이 작품은 NIH / NCI 부여 # (SBE에) R01CA172138에 의해 지원되었다.

동물 주제와 관련된 절차 텔 아비브 대학 기관 동물 관리 및 사용위원회 (IACUC)에 의해 승인되었습니다. 1. 쥐 프로토콜 <ol><li> 준비 <ol><li> 2 나비 21 G 바늘을 정렬합니다. 선택적으로, 바늘의 날카로운 가장자리를 제출하여이 무딘 칼날 나비 21 G 바늘을 준비합니다. </li><li> 중력 (건조) 설정에 적어도 30 분 동안 121 ° C에서 오토 클레이브 멸균 가위 이쌍, 무딘 칼날 집게, 지혈, 치아 조직 겸자 : 수술 도구를 소독. </li><li> 인산염 완충 식염수 (1X PBS) 용액 1 ㎖ 당 30 단위의 헤파린을 첨가하여 PBS 헤파린 (30 유니트 / ㎖)을 준비한다. RT에서 사용합니다. 35 ml의 동물에 따라 필요한 최소한의 볼륨입니다. </li><li> 공기 방울을 방지하기 위해 연동 펌프 라인과 나비 바늘로 헤파린 PBS 스트림. </li></ol></li><li> MP-백​​혈구의 폐와 컬렉션의 관류 <ol><li> 8 %의 이소 플루 란의 과다 복용으로 동물을 안락사. 모니터링 호흡 정지에 의한 안락사를 확인합니다. </li><li> 호흡 중단시 즉시 멸균 가위와 치아 조직 집게를 사용하여 복강와 가슴 충치를 엽니 다. 칼 모양의 과정 복부까지 따라 중간 선 복부 절개를 수행합니다. </li><li> 내부 장기 또는 큰 혈관을 천공하지 않고, 조심스럽게 양쪽의 갈비뼈를 절단 집게를 사용하여 흉골을 들어 올립니다. 흉골에 지혈을 클램프 및 rostrally 심폐 복잡한을 노출 흉곽을 접습니다. </li><li> 심장의 우심실에 나비 바늘을 삽입하고 약 분 이내에 (~ 250g 동물 6 ml)에 주사기로 혈액의 최대 볼륨을 수집합니다. </li><li> 간 (토론 참조)의 후방 관류을 피하기 위해 지혈과 대정맥을 클램프. </li><li> (P)의 유출 관으로 혈액을 함유하는 주사기를 교체하는 동안 우심실 내의 버터 플​​라이 니들 홀드eristaltic 펌프. </li><li> 심실 중격의 침투를 방지 좌심실에 5 ml의 수확 주사기에 연결된 제 2 나비 바늘을 삽입합니다. </li><li> 약 5 ml / 분의 속도로 연동 펌프의 전원을 켜고 조심스럽게 주사기로 혈액으로 오염 관류의 첫 번째 밀리리터를 수집합니다. 관류 색상이 빨간색으로 옅은 어둠에서 꺼질 때까지 계속합니다. (혈액 오염 perfuste 폐기). </li><li> 연동 펌프의 중단없이 빠른 속도로 20 ml의 수확 주사기와 5 ml의 수확 주사기를 교체하고 관류의 더 높은 속도 (최대 7 ml / 분)를 사용하는 폐 관류 20 ㎖를 수집합니다. 진공 형성을 방지하면서 지속적으로 수집 주사기에 관류 흐름을 모니터링한다. </li><li> 연동 펌프의 흐름을 중단. </li></ol></li><li> 백혈구 추출 <ol><li> 400 × g에서 10 분간 원심 분리하여 관류 액. </li><li> 유체를 대기음. </li><li>10 분 동안 400 XG에 10 ml의 PBS 또는 매체, 원심 분리기를 추가하고 유체를 대기음. 두 번이 단계를 반복합니다. PBS 또는 배지의 사용은 시료의 결과적인 사용에 의존한다. </li><li> 10 분 동안 400 XG에 20 ml의 PBS 또는 매체, 원심 분리기를 추가하고 유체를 대기음. </li><li> 시료 사용량 (FACS, PCR 등)에 기초하여, 원하는 농도로 세포를 재구성. </li></ol></li></ol> 2. 마우스 프로토콜 <ol><li> 준비 <ol><li> 2 나비 25 G 바늘을 정렬합니다. 선택적으로, 바늘의 날카로운 가장자리를 제출하여이 무딘 칼날 나비 25 G 바늘을 준비합니다. </li><li> 중력 (건조) 설정에 적어도 30 분 동안 121 ° C에서 오토 클레이브 멸균 가위 이쌍, 무딘 칼날 집게, 지혈, 치아 조직 겸자 : 수술 도구를 소독. </li><li> 1X PBS 용액 1 ㎖ 당 30 단위의 헤파린을 첨가하여 PBS 헤파린 (30 유니트 / ㎖)을 준비한다. RT에서 사용합니다. 25 ml의 동물에 따라 필요한 최소한의 볼륨입니다. </li><li> 헤어 구조 강화공기 방울을 방지하기 위해 연동 펌프 라인과 나비 바늘로 PBS 헤파린을하고있다. </li></ol></li><li> MP-백​​혈구의 폐와 컬렉션의 관류 <ol><li> 8 %의 이소 플루 란의 과다 복용으로 동물을 안락사. 모니터링 호흡 정지에 의한 안락사를 확인합니다. </li><li> 호흡 중단시 즉시 멸균 가위와 치아 조직 집게를 사용하여 복강와 가슴 충치를 엽니 다. 칼 모양의 과정 복부까지 따라 중간 선 복부 절개를 수행합니다. </li><li> 내부 장기 또는 큰 혈관을 천공하지 않고, 조심스럽게 양쪽의 갈비뼈를 절단 집게를 사용하여 흉골을 들어 올립니다. 흉골에 지혈을 클램프 및 rostrally 심폐 복잡한을 노출 흉곽을 접습니다. </li><li> 간 (토론 참조)의 후방 관류을 피하기 위해 지혈과 대정맥을 클램프. </li><li> 오른쪽 VEN으로 연동 펌프의 유출 관에 연결되는 버터 플​​라이 니들을 삽입마음의 tricle하고, 심장, 심실 중격의 avoidingpenetration의 좌심실에 2 ml의 수확 주사기에 연결된 제 2 나비 바늘. </li><li> 약 2 ml / 분의 속도로 펌프를 켜고 관류가 (혈액 오염 perfuste 폐기) 빨간색 옅은 어둠에서 꺼질 때까지 수확 주사기로 혈액 오염 관류의 첫 번째 밀리리터를 수집합니다. </li><li> 연동 펌프의 중단없이 빠른 속도로 10 ml의 수확 주사기 2 ml의 수확 주사기를 교체하고 관류의 더 높은 속도 (최대 4 ml / 분)를 사용하는 폐 관류 10 ㎖를 수집합니다. 진공 형성을 방지하면서 지속적으로 수집 주사기에 관류 흐름을 모니터링한다. </li><li> 연동 펌프의 흐름을 중단. </li></ol></li><li> 백혈구 추출 <ol><li> 400 × g에서 10 분간 원심 분리하여 관류 액. </li><li> 유체를 대기음. </li><li> 400에 10 ml의 PBS 또는 매체, 원심 분리기 추가10 분간 XG 및 유체를 흡인. 이 단계를 세 번 반복합니다. PBS 또는 배지의 사용은 시료의 결과적인 사용에 의존한다. </li><li> 10 분 동안 400 XG에 10 ml의 PBS 또는 매체, 원심 분리기를 추가하고 유체를 대기음. </li><li> 시료 사용량 (FACS, PCR 등)에 기초하여, 원하는 농도로 세포를 재구성. </li></ol></li></ol>

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저자는 공개 아무것도 없어.

본 명세서에있어서, 상기 선택적 수확을 가능 MP-백혈구 고유 인구 공부. 순환 또는 비장 백혈구에 비해 MP 인구 백혈구 개체군의 고유 조성물 높은 활성 수준, 다양한 사이토 카인의 높은 릴리스 및 프로와 항 염증성 사이토 카인 2,3- 높은 mRNA 수준을 특징으로한다. 구체적으로, 우리는 MP-NK 세포는 다양한 표적 세포에 대해 3,4- 순환 NK 세포보다 더 독성이 있고, 인터페론 γ 높은 수?...

폐의 모세 혈관 (즉, marginating - 폐 (MP) 백혈구) (1) 다른 면역 구획 (예를 들어, 순환, 비장, 골수)에서 백혈구에 비해 뚜렷한 백혈구의 구성과 독특한 활성을 나타내는 것으로 나타났다 2-에 부착 백혈구 4. 예를 들어, 순환 및 비장 NK 세포뿐만 아니라, 분화 된 전령 RNA (mRNA의) 레벨 및 프로와 항 염증성 사이토 카인의 분비 증가에 비해 다양한 종양 세포에 대하여 MP-백혈구 전시보다 천연 킬러 (NK) 세포 독성. MP-백​​혈구 순환 백혈구 (50 % 대에 각각 30 %)에 비해 본래 / 적응 면역의 높은 비율을 가지고있는 세포의 조성물은 순환하는 백혈구와 구별된다. 여기에 제시된 방법의 목적은, MP-백혈구의 선택적 수확을 가능하게이 중요하고 독특한 면역 구획 (세포 인구)을 연구하기 위해, 그리고 eluc하는 것입니다이러한 특정 세포에 대한 각종 조작 (예를 들면, 면역 활성)의 효과를 IDATE. 이 독특한 인구의 의미를 이해하기 위해서는, 면역 시스템 세포 매개 면역 (CMI)의 생체 내 기능을 통해 종양 세포, 미세 전이 및 잔여 질병을 순환 제어 할 수 있다는 것이 중요하다. 이 능력은 원발 종양을 제어하는 면역계의 선행 장애에도 불구하고 명확하며, 암 환자 및 동물 모델에서 생체 내 5 충분한 증거에 의해 지원된다. 중요한 것은, 이러한 연구 결과는 6,7가 (세포 독성 분석에 의해 측정) 종종 시험 관내 대부분의자가 종양 세포는 인간과 동물에서 혈액 샘플에서 백혈구를 순환시켜 세포 독성에 내성이보고 생체 연구와 일치한다. 이 차이는 같은 상술과 같은 별개의 백혈구 인구의 생체 내 존재에 기인 할 수있다상술 한 MP 백혈구 인구 및 활성화 NK 세포 (3) 구체적으로는 모집단. 실제로, 순환 및 비장 백혈구에 강한 것으로 밝혀졌다 동계 종양 세포 (MADB106)은, MP-NK 세포 -3,8- 의해 용해되는 것으로 도시 하였다. 따라서 fischer344 (F344) 쥐의 폐에 전이 의심 &#39;NK 내성&#39;MADB106 세포 아니라 순환 또는 일반적 접근 용이성을 부여 다룬다 비장 NK 세포에서 MP-NK 세포에 의해 제어된다. 정제 및 활성 MP-백혈구는 폐 조직의 분쇄 또는 생물학적 분해 (9)를 기반으로 폐에서 백혈구의 표준 수확 방법을 통해 액세스 할 수 없습니다. 우리의 접근 방식은 조직 처리 방법에 비해 두 가지 이점이있다. 먼저, 관류 방법은 선택적으로 다른 폐 실질 간질 세포에서 유래 한 기관지 폐포 비교 예에서 그들을 분리 MP-백혈구 수확tments. 둘째, 더 관류 기술은 분쇄 및 생물학적 처리와 달리 무결성 및 MP-백혈구의 생리적 환경을 유지하는 것은 손상 세포는 그들의 형태를 변경하고, 제조 및 면역 활성을 조절 특히 NK을 억제하는 다양한 인자의 방출을 유도하는 것으로 접근 세포 독성 10. 폐 암 전이 및 각종 감염성 질환의 주요 표적 장기입니다. 모든 순환 악성 세포와 감염된 세포는 변형 및 모세 혈관 내피 세포 및 주민 백혈구와 상호 작용하는 데 필요한 폐 모세 혈관을 통해 전달합니다. 이러한 조건 하에서, 세포의 순환을 용이하게 상주 MP-백혈구 타겟팅 할 수있다. 그것은 따라서이 면역 구획 활성화 백혈구을 가지고 생물학적으로 유리한 것, 그리고 서로 다른 생물학적 실험, 임상 설정에서이 독특한 MP-인구를 연구하는 것이 중요하다. 그 한건데주의하는 가치가있다다양한 생물학적 반응 - 수정하여 stemic 면역 활성화 (예를 들어, polyinosinic-polycytidylic 산 (폴리는 I : C) 또는 유형-C의 CpG 올리고 데 옥시 뉴클레오티드 (의 CpG-C ODN)를), 순환 백혈구 3보다 MP-백혈구 더 활성화하는 것으로 나타났다 8,11.

<table border="0" cellpadding="0" cellspacing="0" width="640">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Autoclud Peristaltic pump</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Butterfly needle</td>
			<td style="width:103px;">OMG</td>
			<td style="width:119px;"></td>
			<td>26G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Butterfly needle</td>
			<td style="width:103px;">OMG</td>
			<td style="width:119px;"></td>
			<td>21G*3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>1 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>2.5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>5 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>10 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Syringe</td>
			<td style="width:103px;">Pic solution</td>
			<td style="width:119px;"></td>
			<td>25 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Blunted-edged forceps</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Scissors</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">hemostat</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tissue forceps</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Heparin sodium porcine mucosa (perservative free)</td>
			<td style="width:103px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

selective harvesting of marginating-pulmonary leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

MP-구획 전시 다른 백혈구 하위 집합 조성물은 백혈구 순환 비교. 유세포 분석을 이용하여, 백혈구 개체군 식별 모두 순환 및 MP 백혈구의 조성물의 특징을 정량화 하였다. 과립구와 림프구는 전방 및 측면 산란을 기반으로 확인되었다. 림프구 내 NKRP-1 세포는 밝은 NK 세포, 단핵구와 같은 T 세포로서 CD3 +, RM1 + 및 CD4 + / CD8 + / CD25로 확...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating - 폐 백혈구의 선택적 수확

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

selective-harvesting-of-marginating-pulmonary-leukocytes

Research

JoVE Journal

Immunology and Infection

5.8K 조회수.  Tel-Aviv University. Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

비디오: Selective Harvesting of Marginating-pulmonary Leukocytes

Isolation of Brain-infiltrating Leukocytes

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine encephalomyelitis virus (TMEV) via a rapid intracranial injection technique and how to purify a leukocyte-enriched population of infiltrating cells from whole brain. Briefly, mice are anesthetized with isoflurane in a closed chamber and are free-hand injected with a Hamilton syringe into the frontal cortex. Mice are then killed at various times after infection by isoflurane overdose and whole brains are extracted and homogenized in RPMI with a Tenbroeck tissue grinder. Brain homogenates are centrifuged through a continuous 30% Percoll gradient to remove the myelin and other cell debris. The cell suspension is then strained at 40 &mu;m, washed and centrifuged on a discontinuous Ficoll-Paque Plus gradient to select and purify the leukocytes. The leukocytes are then washed and resuspended in appropriate buffers for immunophenotyping by flow cytometry. Flow cytometry reveals a population of innate immune cells at the early stages of infection in C57BL/6 mice. At 24 hours post infection, multiple subsets of immune cells are present in the BILs, with an enriched population of Gr1+, CD11b+ and F4/80+cells. Therefore, this method is useful in characterizing the immune response to acute infection in the brain.

A rapid method to obtain infiltrating leukocytes from the murine brain is described. This method utilizes a continuous Percoll gradient and discontinuous Ficoll gradient to select and purify the leukocyte-enriched layer. Isolated leukocytes may then be characterized by flow cytometric measurements.

브레인 - 침투 Leukocytes의 분리

We describe a method for preparing brain infiltrating leukocytes (BILs) from mice. We demonstrate how to infect mice with Theiler's murine ...

연구

면역학 및 감염병학

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

We describe a method for isolation and characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. Infection of susceptible mouse-strains with this parasite strain results in the induction of experimental cerebral malaria, a neurologic syndrome that recapitulates certain important aspects of Plasmodium falciparum-mediated severe malaria in humans 1,2 . Mature forms of blood-stage malaria express parasitic proteins on the surface of the infected erythrocyte, which allows them to bind to vascular endothelial cells. This process induces obstructions in blood flow, resulting in hypoxia and haemorrhages 3 and also stimulates the recruitment of inflammatory leukocytes to the site of parasite sequestration.
Unlike other infections, i.e neutrotopic viruses4-6, both malaria-parasitized red blood cells (pRBC) as well as associated inflammatory leukocytes remain sequestered within blood vessels rather than infiltrating the brain parenchyma. Thus to avoid contamination of sequestered leukocytes with non-inflammatory circulating cells, extensive intracardial perfusion of infected-mice prior to organ extraction and tissue processing is required in this procedure to remove the blood compartment. After perfusion, brains are harvested and dissected in small pieces. The tissue structure is further disrupted by enzymatic treatment with Collagenase D and DNAse I. The resulting brain homogenate is then centrifuged on a Percoll gradient that allows separation of brain-sequestered leukocytes (BSL) from myelin and other tissue debris. Isolated cells are then washed, counted using a hemocytometer and stained with fluorescent antibodies for subsequent analysis by flow cytometry.
This procedure allows comprehensive phenotypic characterization of inflammatory leukocytes migrating to the brain in response to various stimuli, including stroke as well as viral or parasitic infections. The method also provides a useful tool for assessment of novel anti-inflammatory treatments in pre-clinical animal models.

Isolation and Analysis of Brain-sequestered Leukocytes from Plasmodium berghei ANKA-infected Mice

A method for isolation of adherent inflammatory leukocytes from brain blood vessels of Plasmodium berghei ANKA-infected mice is described. The method allows quantification as well as phenotypic characterization of isolated leukocytes after staining with fluorescent antibodies and subsequent analysis by flow cytometry.

에서 뇌 - 분리 된 백혈구의 분리 및 분석<em&gt; Plasmodium berghei</em&gt; ANKA에 감염된 마우스

We describe a method for isolation and  characterization of adherent inflammatory cells from brain blood vessels of P. berghei ANKA-infected mice. ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

인간 백혈구 및 혈소판의 분리 및 평가를위한 프로토콜 : 생물 에너지와 산화 버스트

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Analyzing the Effects of Stromal Cells on the Recruitment of Leukocytes from Flow

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we describe two in vitro &#8220;vascular&#8221; models for studying the recruitment of circulating neutrophils from flow by inflamed endothelial cells. A major advantage of these models is the ability to analyze each step in the leukocyte adhesion cascade in order, as would occur in vivo. We also describe how both models can be adapted to study the role of stromal cells, in this case mesenchymal stem cells (MSC), in regulating leukocyte recruitment.
Primary endothelial cells were cultured alone or together with human MSC in direct contact on Ibidi microslides or on opposite sides of a Transwell filter for 24 hr. Cultures were stimulated with tumor necrosis factor alpha (TNF&#945;) for 4 hr and incorporated into a flow-based adhesion assay. A bolus of neutrophils was perfused over the endothelium for 4 min. The capture of flowing neutrophils and their interactions with the endothelium was visualized by phase-contrast microscopy.
In both models, cytokine-stimulation increased endothelial recruitment of flowing neutrophils in a dose-dependent manner. Analysis of the behavior of recruited neutrophils showed a dose-dependent decrease in rolling and a dose-dependent increase in transmigration through the endothelium. In co-culture, MSC suppressed neutrophil adhesion to TNF&#945;-stimulated endothelium.
Our flow based-adhesion models mimic the initial phases of leukocyte recruitment from the circulation. In addition to leukocytes, they can be used to examine the recruitment of other cell types, such as therapeutically administered MSC or circulating tumor cells. Our multi-layered co-culture models have shown that MSC communicate with endothelium to modify their response to pro-inflammatory cytokines, altering the recruitment of neutrophils. Further research using such models is required to fully understand how stromal cells from different tissues and conditions (inflammatory disorders or cancer) influence the recruitment of leukocytes during inflammation.

The ability of inflamed endothelium to recruit leukocytes from flow is regulated by mesenchymal stromal cells. We describe two in vitro models incorporating primary human cells that can be used to assess neutrophil recruitment from flow and examine the role that mesenchymal stromal cells play in regulating this process.

흐름에서 백혈구의 모집에 기질 세포의 영향 분석

Stromal cells regulate the recruitment of circulating leukocytes during inflammation through cross-talk with neighboring endothelial cells. Here we ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

인간의 산모 - 태아 인터페이스에서 백혈구의 분리

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

산모 - 태아 인터페이스에서 쥐의 조직에서 백혈구의 분리

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

효소 다이제스트 및 그라데이션 분리를 사용하여 마우스 피부에서 침투 백혈구의 분리

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-간 백혈구의 선택적 수확

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Lipopolysaccharide 취급 마우스 간에 면역 반응 동안 백혈구의 2 광자 Intravital 이미징

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

T 세포 수용 체 신호를 통해 화학 억제제 라이브러리의 심사의 중재자의 식별

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...