우리는 우수한 기술 지원 나딘의 Wambsganss 감사합니다. 이 작품은 CAG에 혁신 기금 FRONTIER (하이델베르크 대학)에서 교부금에 의해 도이치 Forschungsgemeinschaft (GL599/1-1)에서 부여 의해 부분적으로 지원되었다

1. 프로토콜 <ol><li> 다음과 같이 버퍼를 준비 <ol><li> PBMC 분리를위한 버퍼를 준비 &quot;버퍼를 씻으&quot;PBS에서 = 0.02 % EDTA (사용 0.5 M EDTA (에틸렌 다이아 민 테트라 초산)).에게 </li><li> 단핵 세포 분리를위한 버퍼를 준비 &quot;MACS 세척 버퍼&quot;= 0.5 % BSA (250 MG) + 2 mM의 EDTA (에틸렌 다이아 민 테트라 초산) (200 μL) + PBS (50 ML). 드가 버퍼입니다. </li><li> &quot;FACS 버퍼&quot;PBS의 = 10 % FCS와 &quot;고정 버퍼&quot;PBS에서 = 1 % PFA : 세포의 FACS 염색 및 저장을위한 버퍼를 준비합니다. </li></ol></li><li> 팔뚝 정맥에서 30 ML 전체 혈액을 그립니다. 항응고제로 EDTA를 사용합니다. </li><li> 다음과 PBMC (histopaque)를 분리 : <ol><li> 두 멸균 50 ML 튜브 (노 폴리 스티렌) 준비합니다. </li><li> PBS 1:1 2 단계에서 혈액을 희석. </li><li> + 25 ML 전체 혈액 / PBS 튜브 당 25 ML의 histopaque를 추가합니다. histopaque 혈액이 혼합하지 않도록주의하십시오. </li><li> RT, 아니 브레이크 30 분 400 XG에 원심 분리기. </li><li> 플라즈마 및 폐기를 대기음. </li><li> 깨끗한 50 ML 욕조에 불투명 인터페이스와 피펫을 대기음전자. </li><li> PBS에 0.​​02 % EDTA (에틸렌 다이아 민 테트라 초산) 20 ML을 추가합니다. </li><li> 4 ℃에서 5 분 250 XG에 원심 분리기 </li><li> 상층 액을 버린다. </li><li> PBS에 0.​​02 % EDTA (에틸렌 다이아 민 테트라 초산) 10 ML을 추가합니다. </li></ol></li><li> 다음과 같이 셀 수 : <ol><li> 10 초 동안 소용돌이로 교반하여 잘 섞는다. </li><li> 200 μl의 세포 현탁액 + 300 μL PBS에 0.​​02 % EDTA (에틸렌 다이아 민 테트라 초산) + 판 블루 (200-500 cells/10 사각형, 그렇지 않으면 희석 배수를 변경)의 500 μL를 가져 가라. </li></ol></li><li> 다음과 같이 단핵구의 음 분리를 수행합니다 : <ol><li> 10 분, 120 X g에 대해 12 단계 3.10에서 얻은 세포를 원심 분리기. 상층 액을 버린다. </li><li> 10 PBS의 ML + 0.02 % EDTA (에틸렌 다이아 민 테트라 초산), 10 분, 120 X g 용 원심 분리기를 추가하여 세포 펠렛 배를 씻으십시오. </li><li> 1 ML 10X PBS를 추가, 3 초 동안 10 ML 멸균 물을 추가합니다. </li><li> PBS + 0.02 % EDTA로 한번 더 씻어 10 분, 120 X g을 원심 분리기. </li><li> 원심 분리하는 동안 계산합니다. </li><li> 5 × 10 7 / ㎖에서 EasySep 버퍼에 희석. </li><li> 50 ML / μL 단핵구 농축 수탉 추가꼬리. </li><li> 4에서 10 분 동안 품어 ° C. </li><li> 30 초 동안 소용돌이 구슬. </li><li> 50 μL / ㎖ 구슬을 추가합니다. </li><li> 4에서 5 분 품어 ° C. </li><li> 2.5 ML의 볼륨을 완료하기 EasySep 버퍼에 채운다. 이 솔루션은 지금 갈색 나타납니다. </li><li> 자석에 넣습니다. </li><li> RT에서 2.5 분을 기다립니다. 이 시간 동안 자기 비드에 결합 이외의 단핵 세포는 관 벽으로 이동하고 거기 찌를 것이다. </li><li> 신선한 멸균 튜브에 구속력 단핵 세포로 버퍼를 붓는다. 이 솔루션은 하얗게 보인다. </li><li> PBS + 0.02 % EDTA 한 번 씻어 10 분, 120 X g 원심 분리기. </li></ol></li><li> 0.5 × 10 6 / ㎠의 밀도에 플라스틱 접시 또는 다중 벽 플레이트 플레이트 세포. 문화 미디어 / 1 X 10 6 세포의 1 ML을 추가합니다. </li><li> 6-14일에 대한 관심의 조건 하에서 배양 세포. </li><li> 새로운 미디어 (자세한 내용은 표 1 참조) 미디어의 50 %를 교체하여 3 일 후에 미디어를 변경합니다. </li><li>mRNA의 분리, 유동 세포 계측법, 웨스턴 블롯 등을위한 6 일 수확 세포 후. </li></ol>

<ol>
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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+intraplaque+hemorrhage+evokes+a+novel+atheroprotective+macrophage+phenotype.">Coronary intraplaque hemorrhage evokes a novel atheroprotective macrophage phenotype.</a> American Journal of Pathology. 174, 1097-1108 (2009).</li><li>Cho, H. 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M., Shiffman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidized+low+density+lipoprotein+exposure+alters+the+transcriptional+response+of+macrophages+to+inflammatory+stimulus.">Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus.</a> Journal of Biological Chemistry. 276, 45729-45739 (2001).</li><li>Qin, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+THP-1+cells+as+a+model+for+mimicking+the+function+and+regulation+of+monocytes+and+macrophages+in+the+vasculature.">The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature.</a> Atherosclerosis. 221, 2-11 (2012).</li><li>Daigneault, M., Preston, J. A., Marriott, H. M., Whyte, M. K. B., Dockrell, D. 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저자는 이익 상충을 공개 할 필요가 없습니다.

단핵구 유래 대 식세포는 타고난 면역 체계의 주요 세포 유형을 나타냅니다. 그들은 죽상 경화증이나 암 2을 (를) 포함하여 많은 염증성 질환에 중요한 역할을한다. 따라서, 대식 세포 생물학을 공부하는 많은 질병의 병태 생리에 대한 우리의 지식을 높이기 위해 필수적이며, 새로운 치료법의 개발을 허용 할 수 있습니다. 많은 연구는 마우스 모델의 과발현으로 적용?...

단핵구 유래 대 식세포는 타고난 면역 시스템의 중요한 세포 구성 요소를 나타내는 많은 급성 또는 만성 염증 과정 1에 기여한다. 대식 세포는 동맥 경화증이나 암이 같은 여러 염증성 질환에 중요한 역할을한다. 대식 세포 가소성의 높은 수준을 보여주고 지역 micromilieu 3에 따라 서로 다른 표현형을 가정 할 수 있습니다. 따라서, 대식 세포의 분화와 이질성을 연구하는 많은 질병의 병태 생리에 대한 지식을 향상을 위해 필수적이며, 새로운 치료 표적과 새로운 치료법 개발의 식별을 허용 할 수 있습니다. 많은 경우에, 쥐 모델은 특정 질병의 병태 생리를 조사하는 데 사용됩니다. 그러나 마우스 모델을 사용하여 대식 세포 생물학을 공부하는 것은 몇 가지 단점이 수반된다 : (1) perip에서 백혈구 집합 숫자의 비율 (즉, 단핵구와 과립구)쥐 또는 인간의 heral 피가 상당히 쥐와 인간의 병태 생리에서 단핵 세포의 서로 다른 역할을 제안 다릅니다. (2) 건강과 질병 4시 그 기능에 상당한 차이를 제안 쥐와 인간의 말초 혈액 단핵 세포 사이의 유전자 발현에 상당한 차이가 있습니다. (3) 인간의 상황에 마우스 모델에서 연구 결과의 전송은 다소 어렵게, 인간의 골수 세포에서 존재하지 않는 쥐 단핵구와 대식 세포 (F4/80, LYC, 등.)를 식별하는 데 사용되는 마커의 번호입니다. 따라서, 인간의 질병에있는 대식 세포의 분화와 이질성에 대한 우리의 이해를 높이기 위해, 우리는 인간 대 식세포와 협력 모델을 사용하도록해야합니다. 그러므로, 우리는 여기에서 사용하기 쉬운 다른 대식 세포 PO의 결과 다양한 조건 하에서 체외에서 인간의 단핵구 유래 대 식세포의 연구를 할 수 있습니다 인간의 기본 대식 세포 생성의 모델을 설명larization 유형. 여러 연구에서, 우리는 대식 세포 생물학, 5-7 인간의 죽상 경화증에 대한 잠재적 인 관련성을 분석하는 단핵구 파생 된 인간의 기본 대식 세포의 체외 모델을 사용했습니다. 비록 완전히 사후 얻은 동물이나 인간의 조직 실험을 교체하지, 여기에 설명 된 모델은 질병의 메커니즘과 다양한 인간의 질병에 매우 관련이있을 수 치료 표적의 식별 및 유효성 검사를 할 수 있습니다.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube (sterile)</td>
			<td>Fisher</td>
			<td>055398</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>D-PBS (1X), liquid (no calcium or magnesium)</td>
			<td>Invitrogen</td>
			<td>14190-250</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>T9285</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A-8806</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Invitrogen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Human Monocyte Enrichment Kit</td>
			<td>StemCell Technologies</td>
			<td>19059</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Fisher</td>
			<td>352008</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Macrophage-SFM (1X)</td>
			<td>Invitrogen</td>
			<td>12065-074</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P-4458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Nutridoma-SP</td>
			<td>Roche</td>
			<td>11011375001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>human M-CSF 10 &mu;g</td>
			<td>Peprotech</td>
			<td>300-25</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell Culture Plates 6-well</td>
			<td>Fisher</td>
			<td>07-200-80</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

an in vitro model to study heterogeneity of human macrophage differentiation and polarization

단핵구 유래 대 식세포는 타고난 면역 시스템의 중요한 세포 유형을 나타냅니다. 대식 세포 생물학을 공부 마우스 모델 생쥐와 인간 단핵구 유래 대 식세포 사이의 표현형과 기능의 차이에서 고통. 그러므로, 우리는 여기에 인간의 기본 대식 세포를 생성하고 공부하는 체외 모델을 설명합니다. 간단히, 팔뚝 정맥에서 도출 말초 혈액의 밀도 기울기 원심 분리 한 후, 단핵구는 부정적인 자기 비드 분리하여 말초 혈액 단핵 세포에서 격리됩니다. 이 단핵구는 다음 대식 세포의 분화 또는 편광의 다른 유형을 유도하기 위해 특정 조건 하에서 육일 동안 배양한다. 모델은 사용하기 쉽고 마우스와 인간 사이의 종 별 차이로 인한 문제를 우회. 또한, 그것은 불후의 세포 라인의 사용을보다 생체 조건에 가깝다. 결론적으로, 여기에 설명 된 모델은 macrophag 연구에 적합하다전자 생물학, 질병 메커니즘과 새로운 치료 표적을 식별합니다. 비록 완전히 사후 얻은 동물이나 인간의 조직 실험을 교체하지, 여기에 설명 된 모델은 질병의 메커니즘과 다양한 인간의 질병에 매우 관련이있을 수 치료 표적의 식별 및 유효성 검사를 할 수 있습니다.

위에서 설명한 프로토콜을 사용하여, 우리는 정기적으로 25.1 X 10 6 ± 2.2 × 10 6 monocytes/100 ML의 피를 (26 독립적 인 실험에서 평균 ± 표준 오차, 그림 1A) 구하십시오. CD14에 대한 유세포 염색에 의해 결정 단핵구 순도는 정기적으로 95 % 이상 (97.1 ± 0.4 %, 3 독립적 인 실험 그림 1B에서 평균 ± 표준 오차)입니다. 판 블루 염색에 의해 결정 갓 고립 된 단핵구 세포 ?...

Watch this Scientific Journal Video about An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization at JoVE.com

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

단핵구 유래 대 식세포는 타고난 면역 시스템의 중요한 세포입니다. 여기, 우리는 사용하기 쉬운 설명<em&gt; 체외에서</em이 세포를 생성하는&gt; 모델. 기울기 원심 분리, 부정적인 비드 격리 및 특정 세포 배양 조건 사용하여 단핵구 유래 대 식세포는 표현형 및 기능 연구에 생성 할 수 있습니다.

<em&gt; 체외</em인간 대식 세포의 분화와 양극화의 이질성을 연구&gt; 모델

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

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Immunology and Infection

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

18.6K 조회수.  University of Heidelberg. 단핵구 유래 대 식세포는 타고난 면역 시스템의 중요한 세포입니다. 여기, 우리는 사용하기 쉬운 설명 체외에서 모델. 기울기 원심 분리, 부정적인 비드 격리 및 특정 세포 배양 조건 사용하여 단핵구 유래 대 식세포는 표현형 및 기능 연구에 생성 할 수 있습니다.

비디오: An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

<em&gt; 체외에서</em공부하기&gt; 공동 감염 모델<em&gt; Plasmodium falciparum</em인간의 기본 단핵구 파생 면역 세포에서&gt; - HIV-1 상호 작용

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

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면역학 및 감염병학

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

골수 유래 대 식세포를 사용하여 대식 세포 분극 조사

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

설립<em&gt; 체외</em&gt; 시스템의 세포 내 행동을 연구하는<em&gt; 칸디다 글라 브라</em&gt; 인간 THP-1 식세포에

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) infections present a broad spectrum of disease severity, ranging from mild infections to life-threatening bronchiolitis. An important part of the pathogenesis of severe disease is an enhanced immune response leading to immunopathology.	Here, we describe a protocol used to investigate the immune response of human immune cells to an HRSV infection. First, we describe methods used for culturing, purification and quantification of HRSV. Subsequently, we describe a human in vitro model in which peripheral blood mononuclear cells (PBMCs) are stimulated with live HRSV. This model system can be used to study multiple parameters that may contribute to disease severity, including the innate and adaptive immune response. These responses can be measured at the transcriptional and translational level. Moreover, viral infection of cells can easily be measured using flow cytometry. Taken together, stimulation of PBMC with live HRSV provides a fast and reproducible model system to examine mechanisms involved in HRSV-induced disease.

An In vitro Model to Study Immune Responses of Human Peripheral Blood Mononuclear Cells to Human Respiratory Syncytial Virus Infection

Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.

Human respiratory syncytial  virus (HRSV) infections present a broad spectrum of disease severity, ranging  from mild infections to life-threatening ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

생체 외 및 생체 내 모델에서 혈관 벽에서 흐름 조건에 세균 부착을 연구하기 위해

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

<em&gt; 체외</em&gt; HIV 공동 감염의 맥락에서 말라리아에 면역 반응을 측정하기위한 모델

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

를 사용하여 림프구 혈관 외로 유출 분석<em&gt; 체외</em&gt; 인간의 혈액 - 뇌 장벽의 모델

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

This protocol describes the fabrication of optical-quality glass surfaces adsorbed with compounds containing long-chain hydrocarbons that can be used to monitor macrophage fusion of living specimens and enables super-resolution microscopy of fixed specimens.

대 식 세포 융합 연구 품질 광학 유리 표면 조작

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging ...

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor (GM-CSF)-producing T Helper (THGM) Cells

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly secretes GM-CSF without producing interferon (IFN)&#947; or interleukin (IL)-17 and is found to play an essential role in the autoimmune neuroinflammation. A method of isolation of naive CD4+ T cells from a single-cell suspension of splenocytes and THGM cell generation from naive CD4+ T cells would be a useful technique in the study of T cell-mediated immunity and autoimmune diseases. Here we describe a method that differentiates mouse naive CD4+ T cells into THGM cells promoted by IL-7. The outcome of the differentiation was assessed by the analysis of the cytokines expression using different techniques, including intracellular cytokine staining combined with flow cytometry, a quantitative real-time polymerase chain reaction (PCR), and enzyme-linked immunosorbent assays (ELISA). Using the THGM differentiation protocol as described here, about 55% of the cells expressed GM-CSF with a minimal expression of IFN&#945; or IL-17. The predominant expression of GM-CSF by THGM cells was further confirmed by the analysis of the expression of GM-CSF, IFN&#945;, and IL-17 at both mRNA and protein levels. Thus, this method can be used to differentiate naive CD4+ T cells to THGM cells in vitro, which will be useful in the study of THGM cell biology.

In Vitro Differentiation of Mouse Granulocyte-macrophage-colony-stimulating Factor GM-CSF-producing T Helper THGM Cells

Here, we present a protocol to differentiate murine granulocyte-macrophage-colony-stimulating-factor-producing T helper (THGM) cells from naive CD4+ T cells, including isolation of naive CD4+ T cells, differentiation of THGM, and analysis of differentiated THGM cells. This method can be applied to studies of the regulation and function of THGM cells.

생체 외에서 마우스 Granulocyte 대 식 세포 콜로 니 자극 인자 (GM-CSF)-T (THGM) 도우미 셀 생산

The granulocyte-macrophage-colony-stimulating factor (GM-CSF)-producing T helper (THGM) cell is a newly identified T helper cell subset that predominantly ...

An Immunohistopathologic Study to Profile the Folate Receptor Beta Macrophage and Vascular Immune Microenvironment in Giant Cell Arteritis

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis and occlusive symptoms of headaches, stroke or vision loss. Macrophages and T-helper lymphocytes infiltrate the vascular wall and produce a pro-inflammatory response that lead to vessel damage and ischemia. To date, there is no GCA biomarker that can monitor disease activity and guide therapeutic response.
Folate receptor beta (FRB) is a glycosylphosphatidylinositol protein that is anchored on cell membranes and normally expressed in the myelomonocytic lineage and in the majority of myeloid leukemia cells as well as in tumor and rheumatoid synovial macrophages, where its expression correlates with disease severity. The ability of FRB to bind folate compounds, folic acid-conjugates and antifolate drugs has made it a druggable target in cancer and inflammatory disease research. This report describes the histopathologic and immunohistochemical methods used to assess expression and distribution of FRB in relation to GCAimmunopathology.
Formalin-fixed and paraffin-embedded temporal artery biopsies from GCA and normal controls were stained with Hematoxylin and Eosin to review tissue histology and identify pathognomonic features.Immunohistochemistry was used to detect FRB, CD68 and CD3 expression. A microscopic analysis was performed to quantify the number of positively stained cells on 10 selected high-power-field sections and their respective locations in the arterial wall.
Lymphohistiocytic (LH) inflammation accompanied by intimal hyperplasia and disrupted elastic lamina was seen in GCA with none found in controls. The LH infiltrate was composed of approximately 60% lymphocytes and 40% macrophages. FRB expression was restricted to macrophages, comprising 31% of the total CD68+ macrophage population and localized to the media and adventitia. No FRB was seen in controls.
This protocol demonstrated a distinct numerical and spatial pattern of the FRB macrophage relative to the vascular immune microenvironment in GCA.

The protocol illustrates the use of histopathologic examination and immunohistochemistry to profile the folate receptor beta macrophage and its relationship with the total immune cell infiltrate in temporal artery biopsies in giant cell arteritis.

Folate 수용 체 베타 대 식 세포와 거 대 세포 동맥에서 혈관 면역 Microenvironment를 프로 파일링 하는 Immunohistopathologic 연구

Giant cell arteritis (GCA) is a chronic immune-mediated disease of medium-to-large sized arteries that affects older adults. GCA manifests with arthritis ...