이 작품은이 자금을 지원 한 보조금 PRB-009 CS와 Secretaría Nacional 드 Ciencia Y Tecnología (SENACYT), 파나마에서 LC에 박사 장학금합니다.

프로토콜의 모든 단계는 centrifugations을 제외하고, 샘플 무균 유지하는 후드 안에 수행해야합니다. 1. P.의 하순 무대 절연 falciparum에 감염된 적혈구 기생충에 paramagnetism을 수여 hemozoin는, 속으로 일반적인 신진 대사이기 때문에 Plasmodium에 감염된 적혈구의 모든 후반 단계는이 방법으로 분리 할 수​​ 있습니다. 전형적인 문화 2 ~ 4 %의 혈소판 (적색 혈액 세포의 부피 비율)와 1-8%의 parasitemia을 (감염된 비율이 포함되어 있지만, 문화에 높은 parasitemia (3-10%)은이 프로토콜보다 나은 수율을하는 것이 좋습니다 적혈구). Trager와 젠슨 (1)에 의해 설명 된대로 기생충을 배양 할 수 있습니다. <ol><li> 간단한 멀티 일부 설정은 모든 schizont 격리 절차를 조립해야합니다. 이를 위해 LS 열, 자기 MidiMACS 구분 기호와 Miltenyi BioTec에서 맥 MultiStand (그림 1 </STRONG&gt;)를 사용하고 있습니다. 첫째, 금속 스탠드에 자기 분리기를 첨부 한 후 분리기에서 자기 열을 배치합니다. </li><li> 별도의 관 (전역 50 ML conicals 사용), RPMI 1640의 23.2 ML 및 작업 솔루션을 생성 할 7.5 % 나트륨 중탄산염의 750 μl를 섞는다. 폐기를위한 손에 추가 튜브를 가지고 흐름을 통해 수집 기생충 문화의. </li><li> 를 평형에 열을 작업 솔루션의 3 ML를 추가하고 흐름을 통해 튜브에 폐기하십시오. </li><li> P. 8 ML을 추가 컬럼에 falciparum 문화, 즉시 작업 솔루션의 마지막 부분으로 문화에 의해 열을 밖으로 밀어되었는지 확인하는, 수신 튜브는 언 바운드 문화의 복구를 시작하기 위해 수집 하나씩 교체됩니다. 이 더 문화를 diluting 방지 할 필요가 있습니다. </li><li> 열을 통해 문화의 흐름이 중단되면, 컬럼의 나머지 적혈구을 밀어 작업 솔루션 1 ML을 추가. </li><li> 단계를 세탁하면 : 열에 작업 솔루션의 또 다른 3 ML을 추가하고 다른 튜브에 흐름을 통해로 저장되는 문화를 diluting 방지하기 위해 &#39;폐기&#39;튜브에 흐르는 액체를 수집합니다. </li><li> 용출 : 3 ML,이 칼럼을 통과 한 후, 스탠드에서 후자를 분리하고 15 ML 튜브의 상단에 배치. </li><li> 다시 열을 작업 솔루션의 3 ML를 추가합니다. 이 단계는 칼럼의 구슬에 갇힌 늦게 무대 기생충에 감염된 적혈구를 elutes. </li></ol> 참고 :이 시점에서, 그리고 필요한 기생충의 양에 따라 원래의 문화의 parasitemia의 한계를 분명히 대상이 컬렉션의 또 다른 라운드 / s를, 시작합니다. 흐름이 정상 상태를 유지로 열이 오랫동안로 활용할 수 있습니다. <ol start="9"><li> 수집 된 기생충의 농도 : 실온에서 150 XG에서 5 분의 eluate를 포함하는 15 ML 튜브를 원심 분리기parasitized 적혈구에서 어두운 펠렛을 형성합니다. </li><li> 아주 작은 샘플을 가지고 (아래 2 단계 참조) 현미경을 통해 농도와 총 생산량을 결정하기 위해 충분한 액체두고 표면에 뜨는을 제거합니다. </li><li> 늦은 무대 기생충의 절연 및 캡처가 만족스럽게 수행하는 경우이 순간에, 신선한 미디어로 다시 수집 문화를 품다. </li><li> 후속 실험에 필요한 볼륨 및 솔루션에 수집 된 기생충을 희석. </li></ol> 2. 순도 및 항복 분석 <ol><li> hemocytometer를 사용하여, 가벼운 현미경으로 계산하여 얻은 기생충의 농도를 확인합니다. 계산하려면, hemocytometer의 커버 슬립에서 수집 된 기생충과 미디어의 혼합의 10 μl를 추가합니다. hemocytometer의 네 당장 자리에 기생충의 수를 세어, 4로 총 수를 나눕니다. 감염된 전자의 농도를 얻기 위해 10 4에 의해 그 수를 모두 곱하면ML 당 rythrocytes. </li></ol> 주 : 믹스의) 농도가 더 많거나 적은 매체를 추가하여 펠렛을 resuspending의 순간에 사용자가 결정합니다. 제안 작업 농도는 μl 당 5.5 X 10 4 schizontes (이 일반적으로 수집 된 기생충에 100-300 μl를 추가하여 달성된다)입니다. 이 농도의 20 μl가 미디어와 적혈구, 전형적인 4 %의 혈소판 문화의 약 1 %의 parasitemia 100 μl 믹스의 최종 볼륨에 추가되어있는 경우 얻을 수 있습니다. b)는 hemocytometer에 따라 계산은 적혈구가 챔버 내부의 샘플 건조로 lysing 시작부터 빨리 할 수​​ 있습니다. 대부분의해야 감염된 적혈구는 비 감염된 사람에서 그들을 차별화 어두운 장소 내부를 표시합니다. <ol start="2"><li> 얇은 층 Giemsa은 10 % 메탄올에 매우 빠르게 슬라이드를 고정을 건조하고, t을 immersing하여 컬렉션의 순결을 평가하기 위해 얼룩을 수행Giemsa의 20 % 솔루션 자락은 증류수로 희석 청바지. 그들이 공기에 의해 건조하고 현미경으로 검사 할 수있는 후 10 분, 대한​​ 슬라이드를 청바지. 잘 70% 위 parasitemia이 달성해야합니다. </li></ol>

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우리는 공개 할 아무 것도 없습니다.

말라리아 기생충 P.의 체외 문화에 falciparum 더 문화의 높은 확산 지점에 감염되지 않은 적혈구의 절반 이상으로 제한된 parasitemia을 나타냅니다. 대부분의 연구 실험을 위해, 감염된 적혈구으로 만 작동하는 것이 바람직하다. 이를 위해 분리 기술은 감염에 따라 문화를 분할 할 필요가 있습니다. 유용한 방법은 감염되지 않은 적혈구 11 두 그룹의 서로 다른 밀도를 이용 차동 centrifugati...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> RPMI 1640 Hepes은 수정 </td><td> 시그마 - 알드리치 </td><td> R4130 </td><td> 10% 인간의 혈청, 2 %의 포도당과 0.2 % 나트륨 중탄산염을 보충 </td></tr><tr><td> MidiMACS 구분 </td><td> 맥 Miltenyi BioTec </td><td> 130-042-302 </td><td></td></tr><tr><td> 맥 MultiStand </td><td> 맥 Miltenyi BioTec </td><td> 130-042-303 </td><td></td></tr><tr><td> LS 열 </td><td> 맥 Miltenyi BioTec </td><td> 130-042-401 </td><td></td></tr><tr><td> Hemacytometer </td><td> Grafco </td><td> Grafco Neubauer 상공 회의소 </td><td> 많은 다른 공급 업체를 통해 찾을 수 있습니다 </td></tr></tbody></table>

separation of plasmodium falciparum late stage-infected erythrocytes by magnetic means

다른 Plasmodium 종과는 달리, P. falciparum은 그 연구 1 용이 실험실에서 배양 할 수 있습니다. 달성 parasitemia는 ≈ 40 % 한도에 도달 할 수 있지만, 탐정은 일반적으로 약 10 %로 비율을 유지합니다. 대부분의 경우 그것은 문화를 풍부하게하고 주어진 실험을 진행하기 위해 감염되지 않은 사람에서 기생충 함유 적혈구를 (적혈구), 분리 할 필요가 있습니다. 때 P. falciparum은 적혈구를 감염 기생충이 저하과 헤모글로빈 2, 3의 피드. 그러나 기생충은 매우 독성 철 함유 haem 잔기 4, 5를 처리해야합니다. 기생충은 haemozoin 6, 7이라는 불활성 크리스탈 폴리머에 haem를 변화함으로써 독성을 도망 간다. 이 철 함유 분자은 ​​그 식품 액포에 저장하고 그 안에 금속이 haem 8 하나에서 다른 산화 상태를 가지고있다. haem의 철의 철 상태ozoin는 감염되지 않은 적혈구의 부재 상자성체의 속성에 수여. 침략 기생충이 성숙에 도달로서, haemozoin의 내용은 P.의 최신 단계에서 더욱 paramagnetism을 준다고하는 9 증가 적혈구 내부 falciparum. 이 상자성체의 속성, P.의 최신 단계에 따라 falciparum 감염 - 적혈구는 자기 구슬이 포함 된 열을 통해 문화를 전달하여 분리 할 수 있습니다. 을 포함하는 열 자석 홀더에 배치 할 때이 구슬은 자석이된다. 흐름을 통해이 포함하면서 paramagnetism로 인해 감염된 적혈구는 다음 대부분의, 감염되지 않은 적혈구 및 기생충들을 포함하는 초기 단계를 들어, 열 안에 갇혀 될 것입니다. 여기, 우리는 좋은 기생충의 생존 능력 (10) 유지 자성 열과 후반 단계 기생충의 인구를 풍부하게 할 수있는 방법에 대해 설명합니다.이 절차를 수행 한 후 무소속의 문화가 남아있는 기생충은 성장을 계속 할 수 있도록 보육에 반환 할 수 있습니다.

그림 2에서, 자기 열을 통과 문화는 전에 표시됩니다 (A)와 절차 후 (B). 화살표가 그림 2A에 감염된 적혈구를 가리키는과 그림 2에 도시 된 바와 같이 하나 두 감염된 적혈구는 보통 100X 배율 필드를 볼 수 있습니다. 전형적인 프로 시저에서 5 % parasitemia (그림 2A)에서 문화로 시작,이 절차의 성능은 일반적으로 97% -100 % ...

Watch this Scientific Journal Video about Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means at JoVE.com

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

hemozoin의 상자성의 특성은 월말 단계를 분리하는 데 사용됩니다<em&gt; Plasmodium falciparum</em문화에 성장&gt; - 감염된 적혈구. 방법은 간단하고 빠르고 기생충의 후속 침습적 기능에는 영향을 미치지 않습니다.

의 분리<em&gt; Plasmodium falciparum</em자기 수단으로&gt; 늦은 단계에 감염된 적혈구

Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ...

separation-plasmodium-falciparum-late-stage-infected-erythrocytes

Research

JoVE Journal

Immunology and Infection

Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

13.8K 조회수.  Instituto de Investigaciones Científicas y Servicios de Alta Tecnología (INDICASAT AIP). hemozoin의 상자성의 특성은 월말 단계를 분리하는 데 사용됩니다 Plasmodium falciparum - 감염된 적혈구. 방법은 간단하고 빠르고 기생충의 후속 침습적 기능에는 영향을 미치지 않습니다.

비디오: Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

Bryostatin의 1/Ionomycin에 의해 종양 반응 T 세포의 생체내 전 확장 및 일반 감마 체인 크린 시토킨 제제

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

연구

면역학 및 감염병학

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

선정 Plasmodium falciparum</em인간 두뇌 내피 세포 Cytoadhesion위한> 기생충

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

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 ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

의 혈소판 응집 중재를위한 간단한 프로토콜<em&gt; 변형체 falciparum의</em자원별로 설정에서&gt; - 감염된 적혈구

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

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. ...

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression of a fluorescent reporter. Here, we highlight an alternative usage of this instrument, for sorting worms according to their degree of colonization by a GFP-expressing pathogen. This new usage allowed us to address the relationship between colonization of the worm intestine and induction of immune responses. While C. elegans immune responses to different pathogens have been documented, it is still unknown what initiates them. The two main possibilities (which are not mutually exclusive) are recognition of pathogen-associated molecular patterns, and detection of damage caused by infection. To differentiate between the two possibilities, exposure to the pathogen must be dissociated from the damage it causes. The wormsorter enabled separation of worms that were extensively-colonized by the Gram-negative pathogen Pseudomonas aeruginosa, with the damage likely caused by pathogen load, from worms that were similarly exposed, but not, or marginally, colonized. These distinct populations were used to assess the relationship between pathogen load and the induction of transcriptional immune responses. The results suggest that the two are dissociated, supporting the possibility of pathogen recognition.

Automated Separation of C. elegans Variably Colonized by a Bacterial Pathogen

The wormsorter facilitates genetic screens in Caenorhabditis elegans by sorting worms according to expression of fluorescent reporters. Here, we describe a new usage:&#160;sorting according to colonization by a GFP-expressing pathogen, and we employ it to examine the poorly understood role of pathogen recognition in initiating immune responses.

의 자동 분리<em&gt; C. 엘레</em&gt; 가변 세균성 병원체에 의해 식민지

The wormsorter is an instrument analogous to a FACS machine that is used in studies of Caenorhabditis elegans, typically to sort worms based on expression ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of free merozoites from the blood by phagocytic cells. However assessing the functional efficacy of merozoite specific opsonizing antibodies is challenging due to the short half-life of merozoites and the variability of primary phagocytic cells. Described in detail herein is a method for generating viable merozoites using the E64 protease inhibitor, and an assay of merozoite opsonin-dependent phagocytosis using the pro-monocytic cell line THP-1. E64 prevents schizont rupture while allowing the development of merozoites which are released by filtration of treated schizonts. &#160;Ethidium bromide labelled merozoites are opsonized with human plasma samples and added to THP-1 cells. Phagocytosis is assessed by a standardized high throughput protocol. Viable merozoites are a valuable resource for assessing numerous aspects of P. falciparum biology, including assessment of immune function. Antibody levels measured by this assay are associated with clinical immunity to malaria in naturally exposed individuals. The assay may also be of use for assessing vaccine induced antibodies. &#160;

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

Measuring antibody function is key to understanding immunity to Plasmodium falciparum malaria. This method describes the purification of viable merozoites, and measurement of opsonization-dependent phagocytosis by flow cytometry.

높은 수익률 정화<em&gt; 변형체 falciparum의</emOpsonizing 항체 분석 실험에 사용하기 위해&gt; Merozoites

Plasmodium falciparum merozoite antigens are under development as potential malaria vaccines. One aspect of immunity against malaria is the removal of ...

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

In Vivo Assessment of Rodent Plasmodium Parasitemia and Merozoite Invasion by Flow Cytometry

The malaria parasite invades and replicates within red blood cells. The accurate assessment of merozoite invasion and parasitemia is therefore crucial in assessing the course of malaria infection. Here we describe a flow cytometry based protocol for the measurement of these parameters in a mouse model of malaria.

유동 세포 계측법에 의해 쥐 말라리아 기생충의 생체 내 평가와 Merozoite 침공

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an ...

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5&#39; RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.&#160; Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.

Methods to Investigate the Regulatory Role of Small RNAs and Ribosomal Occupancy of Plasmodium falciparum

Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.

방법은 작은 RNA를과의 리보솜 점유 규제 역할을 조사하기 위해<em&gt; 말라리아 원충</em

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

생물 발광 영상은 아프리카 트리파노소마 증의 늦은 단계의 감염을 감지하는

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...