우리는 귀중한 토론 이완 - 숙 아, 조앤 모렐로와 앨런 Collmer, 식물 병리학과 식물 - 미생물의 생물학학과, 코넬 대학, 감사합니다. 우리는이 기사에 대한 의견을 제시 Munkvold 감사합니다. 기금은 국립 과학 재단 (National Science Foundation)의 식물 게놈 프로그램에 의해 제공되었다, 보너스 번호 DBI - 0605059 (GBM).

1 부 : 식물의 성장 및 유지 보수 분석에 사용된 Nicotiana benthamiana 공장 약 7 주 된해야합니다. 그들은 모두 겨드랑 지점과 꽃을 제거하기 전에 분석 최소한 4~5일 손질하여야한다. 그것은 그들이 등장 후 식물보다 관리하기 위해 곧 겨드랑 지점을 제거하는 것이 좋습니다. 2 부 : 세균 문화 DAY 1 : <ol><li> 분석에 사용된 모든 변종에 대해 번호판이나 문화가 동시에 시작됩니다. 모나스 종하십시오. 이는 냉동 글리세롤 주식 (표 2)에서 적절한 항생제를 포함하는 KBM 접시에 2 inducers 3 도전 변종, 리크 박테리아를 포함합니다. 박테리아의 소량은 접시의 중앙에 전파해야합니다. 약 18~24시간 30 ° C에서 번호판을 품어. , 30 Agrobacterium 들어, 신선한 플레이트에서 2 ML의 LB에서 액체 문화를 시작하고 250 rpm으로 하룻밤 사이에 성장 ° C. </li></ol> DAY 2 : <ol start="2"><li> 모나스 들어, 박테리아 액체 KBM의 150-200 μl를 추가하고 멸균 유리 확장기를 사용하여 전체 판을 통해 그들을 확산. 다시 인큐베이터에있는 접시를 넣어 다른 20~24시간에 대한 성장하자. 좋은 성장을 나타내는 접시 다음날에 세균 잔디가 있어야합니다. Agrobacterium의 경우, 20 μm의의 acetosyringone로 약 50-10 ML의 LB에서 보조 문화를 시작하고 그것이 250 RPM, 30 하루 20 시간을 기르 ° C. </li></ol> 파트 3 : 분석을위한 준비 식물 DAY 2 : <ol><li> 분석 하루 전에는 식물이 22-24에서 객실로 이동해야 ° C ~ 35~40% RH 지속적인 조명. </li><li> 두꺼운 검은색 마커를 사용하여 직경이 최소 1.5 cm 아르 나뭇잎에 동그라미를 표시합니다. 두 시간 내지 네 동그라미는 잎 당 표시하실 수 있습니다. 동그라미가 잘 간격하고 선호 큰 정맥을 통과해서는 안됩니다. 잘 확장 나뭇잎을 선택합니다. 이전 나뭇잎이나 거친하거나 만지지 두꺼운 있으며 잎의 하단 부분에 동그라미를 표시하지 않도록 나뭇잎을 피하십시오. </li></ol> 마킹 원을 전에 실험에 하루 프로토콜에 필수되지 않습니다. 그러나, 그것은 assayed되는 식물의 큰 숫자가있다면 시간을 절약할 수 있으며, 쉽게 PTI의 유도 도전 예방 접종 사이의 정확한 타이밍을 달성할 수 있습니다. 4 부 : PTI의 유도 3 일 : <ol><li> P. fluorescens와 P. 10 MM MgCl 2 접시에서 세포를 수확 putida. Agrobacterium 들어, 세포를 수집하는 문화를 원심과 10mM MgCl 2 + 10 MM MES 산도 5.6에서 resuspend. MgCl 2 MES 솔루션의 원심 분리와 resuspend를 반복합니다. 600 NM에있는 세포의 광학 밀도 (OD)를 측정합니다. 최종 필요한 값을 표 2에서 설명한대로. 모나스가 드디어 10 MM에 MgCl 2 Agrobacterium을 resuspended되어야 MgCl 2 - MES 솔루션 ODS를 조정합니다. 25 ML의 총 부피는 약 40-50 반점을 예방하기 위해 충분하다. </li><li> 1 ML needleless 주사기를 사용하여 나뭇잎에 미리 표시된 원을에 PTI의 inducers을 침투. 식물이 침투 순서와 침투가 시작되었다 정확한 시간을 적어 두십시오. </li></ol> 제 5 부 : 도전 접종 <ol><li> P. 준비 syringae PV. 토마토 DC3000, 일부 4.1 및 표 2에서 설명한대로 도전 PST DC3000 Δ hopQ1 - 1 PS PV. tabaci. </li><li> 유도가 끝났다고으로 7 시간 유도의 침투 후 식물의 순서에 도전 침투를 수행합니다. 일반적으로, 첫 번째 접종 원의 주변에있는 요점은 두 번째 접종 원의 중심으로 사용할 수 있습니다. 나뭇잎의 여러 장소가있다면 다음 infiltrations가 중복되지 않도록 확인하십시오. </li><li> 정확한 CFU 번호를 확인하기 위해 적절한 항생제를 포함 KBM 또는 LB 접시에 분석에 사용된 모든 문화의 접시 시리얼 dilutions. </li></ol> 6 부 : PTI의 붕괴에 대한 점수 제 5 일 : <ol><li> PST DC3000 맡고 있던 장소에 따라 ETI에 세포 죽음의 모습을 찾아보십시오. 침투의 중복 영역 내부의 세포 죽음은 PTI의 고장을 나타내는 긍정적인 표현형으로 득점한다. </li></ol> 일 7 : <ol start="2"><li> PST DC3000 Δ hopQ1 - 1 또는 PS PV 맡고 있던 장소에 의한 질병에 세포 죽음의 모습을 찾아보십시오. tabaci가 </em&gt;. 일부로 6.1에 설명된 같은 방식으로 긍정적인 표현형에 대한 점수. </li></ol> 컨트롤 식물 (PTI에 대한 타협해서는 안된다는)이 침투의 중복 영역에서 세포 죽음을 표시하기 시작하면, 더 이상 관찰하고 중지합니다. 7 부 : 대표 결과 그림 1은 분석 P.의 결과를 보여줍니다 fluorescens는 과제로 유도 및 PST의 DC3000로 사용되었다. <img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="그림 1" border="0" /> 그림 1. P. fluorescens는 N.에 침투했다 benthamiana 나뭇잎 (블랙 서클)은 PTI를 유도하기 위해 7 시간 후에, 그 자리가 태평양 표준시 DC3000 (흰색 동그라미)와 함께 도전을했습니다. FLS2 위해 입을 식물 P. 지역의 PTI의 세부를 보여주 fluorescens는 세포 죽음으로 볼로, (A)에 침투했다. 침묵되지 않은 컨트롤 식물 PTI (B)의 유도에 의한 중복되는 지역에 세포 죽음을 보여주 없습니다. 빨간색 화살표가 부족하거나 침투의 중복 영역에서 세포 죽음의 존재를 나타냅니다. 사진은 2 일 infiltrations 후에 찍은. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다. <table border="1"><tbody><tr><td> PTI의 유도 </td><td> 세포 죽음 - 도출 도전 </td><td> 세포 사망의 자연 </td><td> 암호 </td></tr><tr><td> 모나스 fluorescens 55 </td><td> 모나스 syringae PV 토마토 (PST). DC3000 6 </td><td> ETI </td><td> PF / DC </td></tr><tr><td> P. putida KT2440 </td><td> PST DC3000 </td><td> ETI </td><td> PP / DC </td></tr><tr><td> P. fluorescens 55 </td><td> PST DC3000 Δ hopQ1 - 1 6 </td><td> 질병 </td><td> Pf/Q1-1 </td></tr><tr><td> Agrobacterium tumefaciens GV2260 </td><td> P. syringae PV. tabaci 11528R </td><td> 질병 </td><td> 농업 / Ptab </td></tr></tbody></table> 표 1 : PTI의 유도 및 세포의 조합 죽는 도출 PTI에 대한 세포 죽음의 분석에 사용되는 미생물. <table border="1"><tbody><tr><td> 박테리아 스트레인 </td><td> 선택 매체 </td><td> 최종 OD 실험에 사용 </td><td> 해당 CFU / ML </td></tr><tr><td> P. fluorescens 55 </td><td> KBM 암피실린 (100 UG / ML) </td><td> 0.5 </td><td> 1 X 10 9 </td></tr><tr><td> P. putida KT2440 </td><td> KBM 암피실린 (100 UG / ML) </td><td> 0.5 </td><td> 1 X 10 8 </td></tr><tr><td> A. tumefaciens GV2260 </td><td> LB Rifampicin (100 UG / ML) </td><td> 0.5 </td><td> 5 X 10 8 </td></tr><tr><td> PST DC3000 </td><td> KBM Rifampicin (100 UG / ML) </td><td> 0.02 </td><td> 2 X 10 7 </td></tr><tr><td> PST DC3000 Δ hopQ1 - 1 </td><td> KBM Rifampicin (100 UG / ML) </td><td> 0.1 100 배 희석 </td><td> 1 X 10 6 </td></tr><tr><td> P. syringae PV. tabaci 11528R </td><td> KBM Rifampicin (100 UG / ML) </td><td> 0.1 100 배 희석 </td><td> 1 X 10 6 </td></tr></tbody></table> 표 2 : 문화 조건과 분석에 사용되는 inoculum 수준. OD와 해당 CFU 수준 spectrophotometers 서로 다른 브랜드 간의 차이가있을 수 있습니다.

<ol>
	<li>Abramovitch, R. B., Anderson, J. C., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+elicitation+and+evasion+of+plant+innate+immunity.">Bacterial elicitation and evasion of plant innate immunity.</a> Nat Rev Mol Cell Biol. 7 (8), 601-60 (2006).</li><li>Jones, J. D., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+immune+system.">The plant immune system.</a> Nature. 444 (7117), 323-323 (2006).</li><li>Zipfel, C., Robatzek, S., Navarro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception.">Bacterial disease resistance in Arabidopsis through flagellin perception.</a> Nature. 428 (6984), 764-764 (2004).</li><li>Oh, H. S., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+resistance+against+bacteria+in+Nicotiana+benthamiana+leaves+is+accompanied+by+reduced+vascular+staining+and+suppressed+by+multiple+Pseudomonas+syringae+type+III+secretion+system+effector+proteins.">Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.</a> Plant J. 44 (2), 348-348 (2005).</li><li>Goodin, M. M., Zaitlin, D., Naidu, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotiana+benthamiana:+its+history+and+future+as+a+model+for+plant-pathogen+interactions.">Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe Interact. 21 (8), 1015-1015 (2008).</li><li>Wei, C. F., Kvitko, B. H., Shimizu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pseudomonas+syringae+pv.+tomato+DC3000+mutant+lacking+the+type+III+effector+HopQ1-1+is+able+to+cause+disease+in+the+model+plant+Nicotiana+benthamiana.">A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.</a> Plant J. 51 (1), 32-32 (2007).</li></ol>

세포 죽음을 기반 분석은 PTI에있는 유전자의 참여를 결정하는 데 사용할 수 있습니다. 예를 들어, 관심있는 유전자에 대한 손실 기능 돌연변이 분석을 사용하여 테스트하실 수 있습니다. 유도와 표 1에서 주어진 도전 예방 접종의 4 조합의 출력, 그것은 하나의 조합이 당신의 식물을 배경으로 표현형을 초래할 수도 있습니다. 우리는 FLS2에 대한 침묵 식물 PF / DC 및 유도 도전 예방 접종 (표 1) Pf/Q1-1 조합 PTI의 세부를 표시 것을 관찰했습니다. 그것은 분석을위한 환경 조건의 일부로 3.1에 ​​설명된 것과 가능한 한 가까운 것을 중요합니다. 낮은 습도는 점수에 대한 분석이 어렵게 너무 빨리 진행하기 위해 세포를 파괴됩니다. 우리 프로토콜에서 설명한 조건이 연구실에 작동하지 않는 경우, 유도 또는 도전의 접종 수준을 변경하십시오. 우리가 짧은 시간 간격은 분석이 통제 공장에서 너무 빨리 무너뜨리는 원인 것으로 나타났습니다하지만 수정할 수 있습니다 또 다른 매개 변수는 유도와 도전 사이의 시간 간격입니다. 우리는 적어도 4-5 식물 실험에 테스트하고 긍정적인 표현형 적어도 3-4 독립적인 실험으로 반복되어야하는 것이 좋습니다.

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<td>MES</td>
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<td>Life Science UV/Vis Spectrophotometer</td>
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assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

자신의 환경에서 잠재적인 병원체를 인식하기 위해, 식물은 패턴 인식 수용체 (PRRs)가 자신의 플라즈마 점막에있는 사용합니다. PRRs는 병원체 - 관련 분자 패턴 (PAMPs)라는 미생물 보존 기능을 인식하고이 감지 효과적으로 비 병원균의 1,2에 의해 식물 조직의 식민을 방지 PAMP - 트리거 면역 (PTI)에 연결됩니다. PTI에서 가장 잘 연구 시스템 FLS2 - 의존 경로 3입니다. FLS2은 박테리아 flagellin의 구성 요소입니다 PAMP flg22를 인식합니다. 성공적인 병원균은 독성 요인이나 PTI을 억제하고 병원균이 질병 1가 발생할 수 있습니다 effectors을 보유하고 있습니다. 차례로 일부 식물 effectors 또는 효과기 - 트리거 면역 (ETI) 2 리드 그들의 활동을 감지 저항 유전자를 보유하고 있습니다. 우리는 아와 Collmer 4 수정 PTI에 대한 세포 사망 기반의 분석을 설명합니다. 분석은 N.의 표준되었습니다 식물 병원체 상호 작용 5 연구의 모델 시스템으로 점차 사용되고 있습니다 benthamiana. PTI는 나뭇잎으로 비 병원성 박테리아 스트레인의 침투에 의해 유발됩니다. 세븐 시간 후에, 그 박테리아 변종도 질병의 원인이나 어느 ETI 원래 침투 영역을 중복 영역으로 가득 활성화됩니다. 최초의 침투에 의해 유도된 PTI는 두 번째 도전 침투에 의한 세포 사망의 모양을 지연 또는 방지하기 위해 수 있습니다. 반대로, 접종의 중복 영역에서 세포 죽음의 모습은 PTI의 고장을 나타냅니다. PTI 도전 예방 접종 inducers의 네 가지 조합 (표 1) 표준화되었습니다. 분석이 아닌 침묵 N.에서 테스트되었습니다 제어 및 PTI를 개발하는 능력에 손상 될 것으로 예측되었다 FLS2에 대한 입막음을 식물로 재직 benthamiana 식물.

Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

Nicotiana benthamiana 공장에서 PTI에 대한 세포 죽음 기반 분석을 설명합니다.

병원체 - 관련 분자 패턴 (PAMP) 트리거된 식물의 내성 (PTI)에 대한 분석

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

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Immunology and Infection

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

27.0K 조회수.  Boyce Thompson Institute for Plant Research. Nicotiana benthamiana 공장에서 PTI에 대한 세포 죽음 기반 분석을 설명합니다.

비디오: Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

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 ...

연구

면역학 및 감염병학

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal&#160;colonization.&#160;The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

In vitro adherence assays can be used to study the attachment of Streptococcus pneumoniae to epithelial cell monolayers and to investigate potential interventions such as the use of probiotics for inhibiting pneumococcal colonization.

를 사용하여 폐렴 구균 식민지에서 생균제의 효과를 조사<em&gt; 체외</em&gt; 준수의 분석

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

전단 응력의 인간 간 사인 내피에서 조건에 백혈구 모집을 연구하는 흐름 접착 분석

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

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 ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

호중구의 분리 및 분석 치료제에 림프종 세포 감도에서 자신의 역할을 확인하는 방법

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

형광 기반의 뉴 라미니다 아제 억제 분석은 항 바이러스제의 뉴 라미니다 아제 억제제 클래스에 인플루엔자 바이러스의 감수성을 평가하기 위해

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

바이러스 성 감염 및 초파리 Melanogaster 상호 작용 호스트-바이러스의 분석의 설립

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

이중 가닥 RNA와 패턴 인식 수용 체 부정적인 감 RNA 바이러스 감염에의 confocal 영상

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;