저자는 그들의 지원은 개발과 immunohistochemistry를 수행하는 기술 미쉘 베인스의 지원뿐만 아니라 박사 페트리 - 핸슨 박사와 티모시 브라운 에얼스를 인식하고 싶습니다. 이 프로젝트는 USDA 크리스 프로젝트 # MISV - 0801310로 및 동물용 의약품의 미시시피 주립 대학에 의해 투자되었다.

<html> 1. 세균 도전 <ol><li> 30 박테리아가 하룻밤 사이에 성장 ° 뇌 심장 주입 용기를 흔들어에서 C. </li><li> 물고기 탱크의 물 순환을 중지하고 100 1 농도 (리터 당 10 ML, 예를 들어)에 국물을 추가합니다. </li><li> 한 시간 부화 후, 남아있는 세균을 제거하기 위해 탱크에 물을 순환을 다시 시작합니다. </li></ol> 2. 샘플링 <ol><li> 샘플링 시간과 기관은 연구의 목적 의존하지만 우리 연구에서, 우리는 다음과 같은 샘플링 포인트 사용 : 1, 6, 16, 24, 36, 48, 60, 72, 96, 1​​10, 125 및 175시간 후 감염 . </li><li> 아가미, 측면 라인, 소장, 비장 따라 사후 근육, 각 시간 점에서 MS222과 샘플 다음과 같은 장기 (샘플 기관의 선택은 감염 과정의 알려진 내용에 기초)가 포함된 물에 침수에 의해 여섯 물고기를 안락사 간, 위, 심장, 머리를 신장하고, 트렁크 신장. </li><li> 샘플링시 즉시 histological 카세트에서 장기 하중, 1 % 포르말린에 두 시간 동안 그들을 담가. </li><li> 그들은 삽입 때까지 남아 두명의 시간 고정 후, 포르말린에서 50 % 에탄올로 카세트를 전송합니다. </li><li> 파라핀은 하룻밤 조직 테크 VIP 3000 조직 프로세서 (사쿠라 테크, 토랜스, 캘리 포니 아주)와 Leica RM2255 microtone을 (Leica 이크로 시스템즈, 배녹번, 일리노 이주)를 사용하여 5μm에서 섹션을 사용하여 장기 포함. </li></ol> 3. Dewaxing 섹션 <ol><li> 디졸브 모든 왁스 수 있도록 약 5 분 맑은 의식으로 가득 얼룩 여물 섹션 젖어. 랙을 들어 여분의 왁스는 용매에서 배수. </li><li> 5 분 분명 라이트의 두 번째 용기에 섹션 젖어. 몇 가지 사용 후, 첫 번째와 두 번째 컨테이너 사이의 첫 번째 컨테이너와 대체의 용매를 교체하십시오. </li><li> 왁스는 용매 제거 100 % 알콜에있는 섹션을 잠그다. </li><li> 100 % 알코올의 두 번째 여물 섹션 젖어. </li><li> 70 % 알콜의 여물 섹션 젖어. </li><li> 알코올의 모든 흔적을 제거하는 수돗물을 실행에있는 섹션을 잠그다. </li></ol> 4. 버퍼 준비 <ol><li> 칼륨은 7.4에 증류수와 산도의 1리터에 8.0 g NaCl, 0.2 g KCl, 1.15 g 나 2 HPO 4 0.2 g KH 2 PO 4 용해하여 생리 버퍼 준비합니다. </li><li> 증류수 100 ML에 PBS 900 ML 추가하여 0.9X에 PBS를 희석. </li><li> 2 ML 마우스 혈청을 추가하여 혈청 차단 준비 200 μl 트리톤 - X 100, 500 μl PBS BSA 0.9X 50 ML에 있습니다. </li><li> 50 MM의 비카보네이트 버퍼 살린 500 ML 증류수 H 2 O에서 2.1 g NaHCO 3 4g NaCl을 해소하고 산도 8.2 titrated에 의해 준비가되어 준비합니다. </li></ol> 5. Immunhistochemistry <ol><li> 0.9X PBS에서 침수로 섹션을 Rehydrate. </li><li> 혈청을 차단 30 분 동안 슬라이드를 품어. </li><li> 상온에서 불투명 습한 챔버에 두 시간 동안 Ed9 (차단 혈청에서 100에 1을 희석)에 섹션을 품어. </li><li> 십분 각 섹션에게 PBS로 4 번 린스. </li><li> 상온에서 불투명 습한 챔버에서, 보조 항체의 50 μl (혈청 차단 500 번 희석 염소 방지 마우스 FitC 표시 항체)에 두 시간 섹션을 품어. </li><li> 비카보네이트 3 시간이 호수 버퍼 섹션 린스. </li><li> 탈이온수에서 잠시 섹션 린스. </li></ol> 6. 설치 <ol><li> 몇 분 동안 어둠 속에서 건조하는 슬라이드를 둡니다. </li><li> 장착은 permafluor를 사용하여 수행됩니다. </li><li> 장착 매체 어두운 시원한 환경에서, 우선적으로 야간, 건조에 둡니다. </li></ol> 7. 현미경 <ol><li> Histological 섹션 형광 현미경으로 관찰하기 위해 준비가되어 있습니다. 필터, 우리의 실험에서, 우리는 트리플 (MCA 텍사스 - 레드 FitC)을 갖춘 올림푸스 BX51 현미경을 사용. 액자 소프트웨어 (MicroFire, 골레타, 캘리포니아)이 사용되었다. </li></ol> 8. 대표 결과 : 물고기 조직의 자동 형광 그렇게 높이 때문에,이 조직은 편리 박테리아에 대한 배경을 제공 트라이 필터 아래에 오렌지가 나타납니다. 이 배경, 개별 FitC 묻은​​ 박테리아는 밝은 녹색 점들로 표시되며, 200X에서 자신의 막대 모양은 (그림 1) 인식할 것입니다. 잘못된 부분은 박테리아의 큰 숫자가 균일하게 슬라이드에있는 것으로 표시되는 경우 (rinsing 단계에서이 문제로 인해) 잘못된 긍정을 구성하실 수 있습니다. <img src="/files/ftp_upload/2687/2687fig1.jpg" alt="그림 1"/> 그림 1. 2 E. 보여주는 근육 섹션 현미경 ictaluri (화살표 A와 B) (200x). igure 2 &quot;/&gt; 그림 2. 실험의 전체 구조

<ol>
	<li>Plumb, J. A., Woo, P. T. K., Bruno, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Diseases and Disorders. , 479-479 (1998).</li><li>Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Pathology. , (2001).</li><li>Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Louisiana State University. , (1997).</li><li>Nusbaum, K. E., Morrisson, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+35S-Labeled+Edwardsiella+ictaluri+into+Channel+Catfish.">Entry of 35S-Labeled Edwardsiella ictaluri into Channel Catfish.</a> Journal of Aquatic Animal Health. 8, 146-146 (1996).</li><li>Karsi, A., Menanteau-Ledouble, S., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bioluminescent+Edwardsiella+ictaluri+for+noninvasive+disease+monitoring.">Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring.</a> FEMS Microbiology Letters. 260, 286-286 (2006).</li><li>Miyazaki, T., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+Edwardsiella+ictaluri+in+channel+catfish,+Ictalurus+punctatus+(Rafinesque).">Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus (Rafinesque).</a> Journal of Fish Diseases. 8, 839-839 (1985).</li><li>Areechon, N., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Edwardsiella+ictaluri+in+Channel+Catfish,+Ictalurus+punctatus.">Pathogenesis of Edwardsiella ictaluri in Channel Catfish, Ictalurus punctatus.</a> Journal of the World Mariculture Society. 14, 249-249 (1983).</li><li>Dumpala, P. R., Lawrence, M. L., Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Edwardsiella+ictaluri.">Proteome analysis of Edwardsiella ictaluri.</a> Proteomics. 9, 1353-1353 (2009).</li><li>Thune, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signature-Tagged+Mutagenesis+of+Edwardsiella+ictaluri+Identifies+Virulence-Related+Genes,+Including+a+Salmonella+Pathogenicity+Island+2+Class+of+Type+III+Secretion+Systems.">Signature-Tagged Mutagenesis of Edwardsiella ictaluri Identifies Virulence-Related Genes, Including a Salmonella Pathogenicity Island 2 Class of Type III Secretion Systems.</a> Applied and Environmental Microbiology. 73, 7934-7934 (2007).</li><li>Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bioluminescence+mutant+screening+strategy+for+identification+of+bacterial+virulence+genes.">High-throughput bioluminescence mutant screening strategy for identification of bacterial virulence genes.</a> Applied and Environmental Microbiology. 75, 2166-2166 (2009).</li><li>Hook, E. W., Mandell, G. L., Douglas, G., Bennett, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practices of Infectious Diseases. , 1700-1700 (1990).</li><li>Ainsworth, J., Capley, G., Waterstreet, P., Munson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+monoclonal+antibodies+in+the+indirect+fluorescent+antibody+technique+(IFA)+for+the+diagnosis+of+Edwardsiella+ictaluri.">Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri.</a> Journal of Fish Diseases. 9, 439-439 (1986).</li></ol>

관심 없음 충돌 선언하지 않습니다.

이 프로토콜 세부 histological 섹션에서 메기 병원체 Edwardsiella ictaluri의 현장 시각화에 대한 기술. 우리의 지식의 최고하려면,이 설명 첫 번째와 같은 프로토콜입니다. 가장 중요한 단계는, 우리의 경험, 잘못된 긍정을 초래할 수로 불충 분한 세척으로 현재 프로토콜의 단계 4.4에서 설명하는 항체의 rinsing입니다. 이 기법의 결과를 해석과 함께 가장 ?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 두뇌 주입 스프 </td><td> 백톤 디킨슨 </td><td> 237200 </td></tr><tr><td> MS222 </td><td> 알젼트 실험실 </td><td></td></tr><tr><td> 전체 마우스 세럼 </td><td> 카펠 </td><td> 55,989 </td></tr><tr><td> 염소 방지 마우스 FitC </td><td> 남부 생명 공학 </td><td> 1010-04 </td></tr><tr><td> Permafluor </td><td> 연구소 비전 </td><td> 타 - 030 - FM </td></tr><tr><td> 염화칼륨 (KCl) </td><td> 시그마 </td><td> P - 4504 </td></tr><tr><td> 트리튼 - X </td><td> 시그마 - 알드리치 </td><td> X100 - 6X500ML </td></tr><tr><td> 소 혈청 알부민 </td><td> 시그마 </td><td> 85040C </td></tr><tr><td> 인산 칼륨 (KH 2 PO 4) </td><td> 시그마 </td><td> P - 5379 </td></tr><tr><td> 중탄산 (NaHCO 3) </td><td> 시그마 </td><td> S - 5761 </td></tr><tr><td> 나트륨 염화물 (NaCal) </td><td> 시그마 </td><td> S - 9625 </td></tr><tr><td> 나트륨 칼륨 이염 (나 2 HPO 4) </td><td> 시그마 </td><td> S - 9390 </td></tr></tbody></table>

use of fluorescent immuno-chemistry for the detection of edwardsiella ictaluri in channel catfish (i. punctatus) samples

Edwardsiella ictaluri 채널 메기 Ictalurus punctatus의 주요 병원체이며 1,2 전 약 30 년간을 발견하고있는 동안, 지금까지,이 작가 &#39;지식의 최고에, 어떤 방법의 현장 시각화에 수 있도록 개발되지 않았습니다 histological 섹션에 박테리아. 세균성 지방화는 플레이트 카운트 3, radiometric 표시된 4, 또는 발광 생물 세균 5를 사용하고 이전의 연구에서 생체내 결정하고있는 동안, 이러한 연구의 대부분은 한 가지 예외를 제외하고 6, 총 장기 수준에서 수행되었습니다. 이 제한은 특히 우려 때문에 E. ictaluri는 복잡한 감염 사이클 1,7을 가지고 있으며, 그것은 독성 요인 8,9 다양한 있습니다. E.의 복잡한 상호 작용 그 호스트 ictaluri 같은 taxonomic 가족에 살모넬라 균 10, 많은 측면에서 비슷합니다. 여기 에인 즈 워스 외 의해 설명되어있는 단클론 항체를 사용하여 Ed9 간접 단백질 histochemistry. 11을 사용하여 박테리아의 탐지를 위해 허용하는 방법을 설명합니다. 간단히 차단 혈청가 아닌 특정 기다리는을 방지하기 위해 파라핀 임베디드 histological 섹션에 적용됩니다. E. 그런 다음 섹션은 기본 항체와 incubated 아르 ictaluri 특정 단클론 항체 Ed9. 초과 항체가 떨어져 씻어서되고 FitC 라벨 이차 항체가 추가됩니다. rinsing 후, 섹션은 형광등 특정 설치 매체로 탑재하고 있습니다. 이것은 E.의 검색 허용 채널 메기 조직의 histological 섹션에 현장에 ictaluri.

Watch this Scientific Journal Video about Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples at JoVE.com

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

여기의 레이블을 허용하는 절차를 설명<em현장에서&gt; Edwardsiella ictaluri</em&gt; 채널 메기에서 histological 섹션<em&gt; Ictalurus punctatus</em&gt; 보조로 기본 및 형광 FitC 표시 항체 단클론 항체와 같은 간접 immunohistochemistry Ed9를 사용합니다. 이것은 형광 현미경을 사용하여 세균의 검출 허용.

의 검출을위한 형광 단백질 화학의 이용 Edwardsiella ictaluri 채널 메기에 ( I. punctatus) 샘플

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

use-fluorescent-immuno-chemistry-for-detection-edwardsiella-ictaluri

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Immunology and Infection

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

10.4K 조회수.  Mississippi State University. 여기의 레이블을 허용하는 절차를 설명 Edwardsiella ictaluri 채널 메기에서 histological 섹션 Ictalurus punctatus 보조로 기본 및 형광 FitC 표시 항체 단클론 항체와 같은 간접 immunohistochemistry Ed9를 사용합니다. 이것은 형광 현미경을 사용하여 세균의 검출 허용.

비디오: Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

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면역학 및 감염병학

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

Oligonucleotide - 결합 형광등 Microspheres를 사용하여 복잡한 임상 및 환경 샘플에서 박테리아의 멀티 플렉스 감지

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

유동 세포 계측법에 의해 주요 면역 세포 모집단과 형광 나노 입자의 상호 작용의 검출

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

T 세포 반응의 평가를위한 형광 대상 배열의 사용<em&gt; 생체 내</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

의 검출을위한 자석 교반기 방법<em&gt; 선모충</em근육 샘플에서&gt; 애벌레

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

전체 혈액 샘플에서 바이러스의 검출, 안전, 신속 하 고 간단한 수동 침대 핵 산 추출

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

살 모 넬 라 종의 심사에 대 한 높은 처리 플랫폼 /Shigella 종

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

멀티플렉스 형광 Immunohistochemical 얼룩, 이미징, 및 림프 종 조직학 샘플 분석

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

VirWaTest, 물 샘플에서 바이러스 검출을 위한 사용 시점 방법

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.