이 작품은 건강과 낭성 섬유증 재단의 국립 연구소에서 교부금에 의해 지원되었다.

1. LPS 추출을위한 박테리아의 작성 <ol><li> 항생제 필요한 경우에 보충 Luria 국물 (LB), 5 ML에서 하룻밤 문화를 시작합니다. 37시 배양기를 흔들어 ° C ~ 200 RPM의 (12-18시간) 야간 문화를 성장. </li><li> LB와 문화 1시 10분을 희석하고 분광 광도계에서 OD 600 독서 가져가라. OD 600 독서를 바탕으로 0.5의 OD 600으로하여 박테리아의 1.5 ML 서스펜션을합니다. </li><li> 펠렛은 10 분 10,600 X g에서 microcentrifuge의 박테리아. 표면에 뜨는를 제거하고 폐기. LPS를 즉시 추출되지 않을 경우 펠렛은 -20 ° C에 저장할 수 있습니다. </li></ol> 2. LPS의 추출 <ol><li> 첫째, 2X SDS 버퍼를 준비합니다. 0.1에서 50 ML의 4% β-메르 캅 토 에탄올 (BME) 4 % SDS의 솔루션 및 20 % 글리세롤 만들기 M 트리스-HCL, 산도 6.8. 솔루션을 염색하는 bromophenol 파란색의 핀치를 추가합니다. 2X을 diluting하여 1X SDS-버퍼 주식을 만들기멸균 증류수 H 2 O에 관련 째깍 1시 1분 이것은 상온에서 저장할 수 있습니다. </li><li> 멸균 증류수 H 2 O에에 DNase 나 RNase와 Proteinase K의 세 별도의 10 밀리그램 / ML 솔루션 만들기 </li><li> 1X SDS-버퍼 200 μl의 단계 1.3에서 pelleted 박테리아를 Resuspend. 펠렛 완전히 천천히 아래로 솔루션을 pipetting 및하여 resuspended되었는지 확인하십시오. 와동하지 마십시오. </li><li> 15 분 동안 물을 욕조에 정지 박테리아를 끓이다. 15 분 동안 상온에서 냉각 솔루션을 허용합니다. </li><li> 2.2에서 준비 DNase I 및 RNase 솔루션 모두 5 μl를 추가합니다. 30 분간 37 ° C에서 샘플을 품어. *이 단계는 선택 사항입니다, nuclease-대우와 비 대우 샘플 사이 LPS의 품질에 최소한의 차이가있다. </li><li> 2.2에서 준비 Proteinase K의 솔루션을 10 μl를 추가합니다. 3 시간 동안 59 ° C에서 샘플을 품어. 시간 제약이있는 경우이 단계는 하룻밤 사이에 수행할 수 있습니다. </li><li> 각 샘플, 광고까지의 D 200 μl는 얼음처럼 차가운 트리스 - 포화 페놀 (트리스 - 포화 페놀를 사용할 수없는 경우에는 수상 포화 페놀은 대신 사용될 수 있습니다.) 튜브가 꽉 닫혀있에 모자, 약 5~10초위한 와동 각 샘플을 확인합니다. </li><li> 가끔 vortexing, 15 분 동안 65 ° C에서 샘플을 품어. 실온에 시원한 잠복기 후, 다음 1 각 샘플로 룸 온도 디에틸 에테르 ML 5로 10 초간 소용돌이를 추가합니다. 그것이 휘발성이기 때문에, 연기 후드에 핸들 에테르를 수행하십시오. </li><li> 10 분 동안 20,600 X g에서 샘플을 원심 분리기. 조심스럽게 원심 분리기에서 샘플을 제거하고 아래쪽에 파란색 레이어를 추출합니다. 상위, 투명 레이어를 사용하지 않아야합니다. 파란 레이어의 소량 뒤에두면 상위 레이어와 오염 것이 바람직합니다. </li><li> 2.9 - 단계 2.7을 반복하여 샘플을 다시 압축을 풉니다. 두 extractions은 일반적으로 충분합니다. 샘플 흐린 나타나는 경우 자세한 extractions는 performe 수 있습니다라. SDS-PAGE로 분리되기 전에 추출된 표본 각 2X SDS-버퍼 200 μl를 추가합니다. 샘플은 8% -15 %는 SDS-polyacrylamide의 젤에서 실행할 수 있습니다. 이 방법을 사용 준비가 LPS의 다섯 데 15 μl는 시각화를 위해 일반적으로 충분합니다. </li></ol> 3. 대표 결과 위와 같이 준비 LPS 샘플 표준 실버 염색법 프로토콜이나 상용 LPS의 염색법 키트를 사용하여 SDS-PAGE에서 분리에 따라 직접 염색법에 의한 시각이 될 수 있습니다. 또는 polyacrylamide 젤에 분리된 LPS는 nitrocellulose 막에게 양도 및 LPS 특정 안티 세라를 사용하여 immunoblotting 서양를 받게됩니다. 이 프로토콜의 경우, 우리는 프로-Q 에메랄드 300 Lipopolysaccharide 젤 얼룩 키트 (분자 프로브)를 사용하고, 제조 업체의 지침을 따라갔다. 프로-Q 에메랄드 300 스테인드 12% SDS-polyacrylamide 겔은 그림 1에 표시됩니다. 각 레인은 DIF에서 준비 LPS의 15 μl를 포함Burkholderia dolosa의 ferent 변종은 낭성 섬유증 환자의 가래 샘플로부터 격리. 사다리 패턴을 banding 다른 LPS, 다른 숫자 올리 고당 코어에 연결된 장치를 반복 O-항원의 반사,이 방법을 사용하여 증거들이, 예를 들어, 레인 1과 6의 샘플이 서로 유사한 banding 패턴을 가지고 (빨간색 박스). <img alt="그림 1" src="/files/ftp_upload/3916/3916fig1.jpg" /> Burkholderia dolosa 임상 분리의 그림 1. 프로-Q 에메랄드 300 스테인드 LPS. 일곱 B.로부터 LPS 이 프로토콜의 설명에 따라 낭성 섬유증 환자의 확산에서 dolosa의 종자는 고립되었다. 열 다섯 μl는 제조 업체의 지침에 따라, 프로-Q 에메랄드 300과 12% SDS-polyacrylamide 젤, 그리고 스테인드에서 분리되었다. LPS 코어는 화살표를 표시하고, O-항원 반복 유사한 banding 패턴을 보여주는 LPS는 빨간색으로 박스가 있습니다. M은 = 분자량 마커.

<ol>
	<li>Raetz, C. R., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipopolysaccharide+endotoxins.">Lipopolysaccharide endotoxins.</a> Annu. Rev. Biochem. 71, 635-700 (2002).</li><li>Valvano, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Export+of+O-specific+lipopolysaccharide.">Export of O-specific lipopolysaccharide.</a> Front. Biosci. 8, 452-471 (2003).</li><li>Pier, 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=Pseudomonas+aeruginosa+lipopolysaccharide:+a+major+virulence+factor,+initiator+of+inflammation+and+target+for+effective+immunity.">Pseudomonas aeruginosa lipopolysaccharide: a major virulence factor, initiator of inflammation and target for effective immunity.</a> Int. J. Med. Microbiol. 297, 277-295 (2007).</li><li>Freudenberg, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+lipopolysaccharide+susceptibility+in+the+innate+immune+response+to+Salmonella+typhimurium+infection:+LPS,+a+primary+target+for+recognition+of+Gram-negative+bacteria.">Role of lipopolysaccharide susceptibility in the innate immune response to Salmonella typhimurium infection: LPS, a primary target for recognition of Gram-negative bacteria.</a> Microbes Infect. 3, 1213-1222 (2001).</li><li>Jann, K., Jann, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysaccharide+antigens+of+Escherichia+coli.">Polysaccharide antigens of Escherichia coli.</a> Rev. Infect. Dis. 9, S517-S526 (1987).</li><li>McCallum, K. L., Schoenhals, G., Laakso, D., Clarke, B., Whitfield, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-molecular-weight+fraction+of+smooth+lipopolysaccharide+in+Klebsiella+serotype+O1:K20+contains+a+unique+O-antigen+epitope+and+determines+resistance+to+nonspecific+serum+killing.">A high-molecular-weight fraction of smooth lipopolysaccharide in Klebsiella serotype O1:K20 contains a unique O-antigen epitope and determines resistance to nonspecific serum killing.</a> Infect. Immun. 57, 3816-3822 (1989).</li><li>Hancock, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+isolates+from+patients+with+cystic+fibrosis:+a+class+of+serum-sensitive,+nontypable+strains+deficient+in+lipopolysaccharide+O+side+chains.">Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains.</a> Infect. Immun. 42, 170-177 (1983).</li><li>Gupta, S. K., Masinick, S., Garrett, M., Hazlett, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+lipopolysaccharide+binds+galectin-3+and+other+human+corneal+epithelial+proteins.">Pseudomonas aeruginosa lipopolysaccharide binds galectin-3 and other human corneal epithelial proteins.</a> Infect. Immun. 65, 2747-2753 (1997).</li><li>Hitchcock, P. J., Brown, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+heterogeneity+among+Salmonella+lipopolysaccharide+chemotypes+in+silver-stained+polyacrylamide+gels.">Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.</a> J. Bacteriol. 154, 269-277 (1983).</li><li>Westphal, O., Jann, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+lipopolysaccharides.">Bacterial lipopolysaccharides.</a> Methods Carbohyr. Chem. 5, 83 (1965).</li><li>Lieberman, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+bacterial+evolution+within+multiple+patients+ties+novel+genes+to+pathogenesis.">Parallel bacterial evolution within multiple patients ties novel genes to pathogenesis.</a> Nat Genet. , (2011).</li></ol>

관심의 어떠한 충돌 선언 없습니다.

우리는 핵산과 단백질 등 다른 세포 구성 요소에서 LPS를 멀리 정화하는 방법을 설명했습니다. 이 방법은 그림 1에 표시된 SDS-PAGE의 젤의 탄수화물의 염색법을 포함한 다양한 시각화 방법, 다수에서 사용할 수있는 고품질의 LPS를 제공합니다. 이 방법은 특정 안티 세라를 사용하여 종자의 다양한 LPS를 혈청형하거나 직접 시각화하여 분리 사이 relatedness을 표시하는 데 사용할 수 있습니다. 예를 들어,...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> DNase 나 재조합, RNase가없는 </td><td> 로슈 </td><td> 04716728001 </td></tr><tr><td> RNase </td><td> 로슈 </td><td> 10109169001 </td></tr><tr><td> Proteinase K </td><td> 어부 </td><td> BP1700 </td></tr><tr><td> 페놀 트리스 - 포화 </td><td> 어부 </td><td> BP1750-100 </td></tr><tr><td> 디에틸 에테르 </td><td> 토마스 과학 </td><td> C313K31 </td></tr><tr><td> 프로-Q 에메랄드 300 Lipopolysaccharide 젤 얼룩 키트 </td><td> 분자 프로브 </td><td> P20495 </td></tr></tbody></table>

purification and visualization of lipopolysaccharide from gram-negative bacteria by hot aqueous-phenol extraction

Lipopolysaccharide가 (LPS) 그람 음성 세균의 외부 세포막의 주요 구성 요소입니다. 그것은 지방질로 구성된 삼자 간의 분자는 외부 세포막에 박혀있는, 셀 1, 2의 표면에서 바깥쪽을 확장하는 핵심 올리 고당 및 반복 O-항원 단위. LPS는 녹농균, 살모넬라균 종, 그리고 대장균 3-5 및 LPS O-항원 구성 양식 종자의 serotyping의 기초의 차이 등 많은 세균 종의 독성 및 pathogenesis에 중요 immunodominant 분자이다. LPS는 감염의 개시에 세포를 호스트 첨부에 참여하고 보완 - 중재 죽이는에서 보호를 제공합니다; LPS 부족 변종은 독성 6-8에 대한 감쇠가 될 수 있습니다. 이러한 이유로, 그것은 특히 임상 분리에서 LPS를 시각화하는 것이 중요합니다. 특정 의해 패턴과 인식을 banding 떠올리 LPSntibodies는 스트레인 lineages를 식별할 수 있도록, 다양한 돌연변이를 특성화하는 데 유용한 도구가 될 수 있습니다. 이 보고서에서 우리는 그람 음성 박테리아 세포에서 LPS의 분리 및 정제를위한 고온 수성 - 페놀 방법을 설명합니다. 이 프로토콜은 멀리 구 짧고 덜 집약적인 추출 방법으로 발생 LPS의 시각화를 방해할 수있는 핵산과 단백질에서 LPS의 추출을 허용합니다. LPS이 방법은 탄수화물 / 당단백질 얼룩 또는 표준 실버 염색법 방법을 사용하여 나트륨 dodecyl 황산 (SDS)-polyacrylamide 겔 전기 영동 (PAGE)와 직접 스테인드으로 구분됩니다 준비. LPS로 방지 세라 많은 저해 수있는 외부 막 단백질이나 다른 antigenic 목표와 교차 반응 반응은 SDS-PAGE로 구분 원유 세포 lysates의 서양 immunoblot에 따라 관찰되는 항체가 포함되어 있습니다. 혼자 원유 세포 lysates의 단백 분해 효소 처리는 항상 이것을 제거하는 효과적인 방법 없습니다이 문서나 다른 시각화 방법을 사용하여 배경. 또한, 이러한 배경을 제거하기 위해 광범위한 단백 분해 효소 처리가 잘 앞서 언급한 방법 중 하나가 해결되지 품질의 LPS로 이어질 수 있습니다. 이러한 이유로, 우리는 Westpahl 및 Jann 10 일부터 맞게 다음과 같은 프로토콜, LPS 추출에 이상적이라고 생각합니다.

Watch this Scientific Journal Video about Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction at JoVE.com

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

우리는 그람 음성 세균의 lipopolysaccharide을 (LPS) 정화를위한 수정된 뜨거운 수성 - 페놀 추출 방법을 설명합니다. 일단 압축을 푼, LPS는 연속적으로 SDS-PAGE에 의해 분석 및 직접 염색법이나 서양 immunoblot에 의해 시각하실 수 있습니다.

핫 수성 - 페놀 추출에 의한 그람 음성 박테리아에서 정화와 Lipopolysaccharide의 시각화

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

purification-visualization-lipopolysaccharide-from-gram-negative

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JoVE Journal

Immunology and Infection

35.0K 조회수.  University of Virginia Health System. 우리는 그람 음성 세균의 lipopolysaccharide을 (LPS) 정화를위한 수정된 뜨거운 수성 - 페놀 추출 방법을 설명합니다. 일단 압축을 푼, LPS는 연속적으로 SDS-PAGE에 의해 분석 및 직접 염색법이나 서양 immunoblot에 의해 시각하실 수 있습니다.

비디오: Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

형광 활성 세포 정렬 (외과)에 의해 특정 세포 인구의 정제

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

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면역학 및 감염병학

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IgY 기술 : 폴리에틸렌 글리콜의 노른자위에서 치킨 항체의 추출 (PEG) 강수량

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

에서 DNA 추출을위한 간단한 Chelex 프로토콜<em&gt; 아노펠레스</em&gt; 종.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

병원체 Vacuoles의 정제에서<em&gt; Legionella</em&gt; - 감염된 Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

MALDI-TOF 질량 분석기를 사용하여 혈액 배양액에서 그람 음성 박테리아의 빠른 확인

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T 세포는 수지상 세포에서 Transinfection에 의해 박테리아를 캡처

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA 정제는 세포 내에서 재배<em&gt; 리스테리아</em&gt; 식세포 세포에

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with respect to how viral members of the microbiome affect subsequent microbe interactions with these virus-infected host cells. The co-habitation of virus with bacteria and fungi is principally present on the mucosal surfaces of the oral cavity and genital tract. Mucosal cells, particularly those with persistent chronic or persistent latent viral infections, could have a significant impact on members of the microbiome through virus alteration in number and type of receptors expressed. Modification in host cell membrane architecture would result in altered ability of subsequent members of the normal flora and opportunistic pathogens to initiate the first step in biofilm formation, i.e., adherence. This study describes a method for quantitation and visual examination of HSV&#39;s effect on the initiation of biofilm formation (adherence) of S. aureus and C. albicans.

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

박테리아와 균류에 의해 바이러스에 감염된 세포에 바이오 필름 개시의 결정

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

절연, 특성화 및 비만 관련 염증에 의해 영향을 받는 조직에서 세포의 정화

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the goal of this protocol is to develop a method to visualize the pathogen invasion in a specific immune compartment of flies, namely hemocytes. Using the method presented here, up to 3 &#215; 106 live hemocytes can be obtained from 200 Drosophila 3rd instar larvae in 30 min for ex vivo infection. Alternatively, hemocytes can be infected in vivo through injection of 3rd instar larvae followed by hemocyte extraction up to 24 h post-infection. These infected primary cells were fixed, stained, and imaged using confocal microscopy. Then, 3D representations were generated from the images to definitively show pathogen invasion. Additionally, high-quality RNA for qRT-PCR can be obtained for the detection of pathogen mRNA following infection, and sufficient protein can be extracted from these cells for Western blot analysis. Taken together, we present a method for definite reconciliation of pathogen invasion and confirmation of infection using bacterial and viral pathogen types and an efficient method for hemocyte extraction to obtain enough live hemocytes from Drosophila larvae for ex vivo and in vivo infection experiments.

Extraction of Hemocytes from Drosophila melanogaster Larvae for Microbial Infection and Analysis

This method demonstrates how to visualize pathogen invasion into insect cells with three-dimensional (3D) models. Hemocytes from Drosophila larvae were infected with viral or bacterial pathogens, either ex vivo or in vivo. Infected hemocytes were then fixed and stained for imaging with a confocal microscope and subsequent 3D cellular reconstruction.

미생물 감염 및 분석에 대 한 초파리 melanogaster 애벌레에서 Hemocytes의 추출

During the pathogenic infection of Drosophila melanogaster, hemocytes play an important role in the immune response throughout the infection. Thus, the ...

핫 수성 - 페놀 추출에 의한 그람 음성 박테리아에서 정화와 Lipopolysaccharide의 시각화

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