저자는 모기가 양육을 위해 존 프라이와 벤 피터슨 감사하고 싶습니다. 우리는 또한 hemocyte micrographs와 관련하여 도움이 크리스틴 데이비스와 게리 Radice 감사드립니다. 이 연구는 리치몬드 예술 &amp; AA Qayum와 A. Telang에 교수 부여하는 과학 여름 교제 대학에 의해 투자되었다.

1. hemocyte 컬렉션 사전에 준비 <ol><li> (; 98 MM NaOH, 186 MM NaCl, 1.7 MM EDTA (에틸렌 다이아 민 테트라 초산), 41 MM 구연산 산성, pH를 4.5 항응고제) 60% 슈나이더의 중간 10 % 태아 소 혈청 (FBS) 및 30 % 구연 산염 버퍼의 구성된 hemolymph의 희석제 솔루션을 준비 11. -20 ° C.에 저장할 수 있습니다 1.0 ML aliquots 만들기 (자료 참조) 한 번만 각 나누어지는을 사용합니다. </li><li> 유리 에칭 도구를 사용하여 한쪽 끝에 1cm 직경의 원을 채점하여 유리 현미경 슬라이드 (75 X 25 X 1mm)를 준비합니다. 또는 두 사전 에칭 원을과 현미경 슬라이드 (예 : 피셔 과학 또는 EMS 소스) 구입할 수 있습니다. 모기 당 하나의 유리 슬라이드가 필요합니다. </li><li> 유리 바늘 세트를 (자료 참조) 준비는 다음과 같은 제안 설정 (셔터 인 스트 루먼트, 모델 P - 87)에 따라 가져온 : <table border="1"><tbody><tr><td> 열 램프 5 </td></tr><tr><td> 당기 : 45 </td></tr><tr><td> 벨 : 75 </td></tr><tr><td> 시간 : 175 </td></tr><tr><td> 압력 580 </td></tr></tbody></table> 이 풀러의 제안 설정을 사용하여, 우리는 약 3mm의 생크 길이와 전체 길이 50​​0mm 주위 바늘을 구축. 미리 뽑아 바늘도 (예 : Tritech 연구) 구입할 수 있습니다. </li></ol> 2. hemocyte 컬렉션 사전에 microinjector의 준비 <ol><li> 제조 업체의 지침에 따라 microinjector 니들 홀더에 뽑아 바늘을 삽입합니다. Backfill는 microinjector (자료 참조) 튜브, 주사기 및 제조 업체의 지침에 따라 광유와 유리 바늘을 뽑아. </li></ol> 3. hemocyte 컬렉션 사전에 여성 모기의 준비 <ol><li> 얼음과 접촉 수있는 작은 고체 케이지 용기에 15 성인 여성을 대기음. 깊은만큼 얼음에 모기와 함께 젖어 케이지는 케이지 모든 측면의 전체 높이 때문에 얼음과 접촉하고 있습니다. 이것은 모든 모기가 추위에 노출 될 수 있도록합니다. 약 8 분 또는 때까지 차가운 마취의 모기들은 더 이상 이동되지 않습니다. </li><li> 우울증 슬라이드에 한 모기를 놓습니다. 이 슬라이드는 주사에 대한 사이트로 될 것입니다. </li></ol> 4. Hemocyte 모음 <ol><li> 가져온 유리 바늘로 hemolymph의 희석제 12 μl를 타고 일곱 번째와 여덟째 복부 세그먼트 사이 모기에 솔루션을 주입하는 microinjector을 설정합니다. 약 3 모기가 차지하는 총 12 μl 볼륨 3-3.5 μl로 주입 수 있습니다. 주입이 수행되는 동안 모기는 부드럽게 포셉와 장소에서 개최한다. 희석액의 작은 볼륨 주입되는 미네랄 오일을 방지하기 위해있는만큼 바늘의 끝에 유지됩니다. 주사 후 복부는 &quot;가득&quot;또는 부풀어 보일 것입니다. </li><li> 장소는 5 분 복구 얼음에 별도의 깨끗한 용기에 모기를 주입했다. 모기 내부 항응고제 희석제 솔루션의 부화는 내부 조직을 준수 hemocytes을 이동시키다 도움이 될 것입니다. 평균 3-5 신선한 모기는 각 5 분 복구 기간 동안 주입 수 있습니다. </li><li> 복구, 다리에서, 싹둑, 날개, 그리고 마이크로 가위를 사용하여 한 번에 하나씩 각 모기 하나의 복부 (여덟째 세그먼트에서)의 팁 5 분 후에. 이것은 얼음에 게재 복구 컵에서 수행되어야합니다. 다리와 날개를 잘랐다 수집 슬라이드로 전송되는 저울의 가능성을 줄여줍니다. 참고 : 다리를 자르고 날개도 흉부 첨부 파일 사이트에 가까이 또는 hemolymph 탈출하기 위해 다른 구멍을 만드는 위험하지 않습니다. Hemolymph은 복부의 끝에 만들어진 구멍을 통해 수집해야합니다. </li><li> 70 % 에탄올 한 유리 에칭 현미경 슬라이드를 세척하고 kimwipe로 건조. 위치 하나는 에칭 원의 바깥쪽 가장자리에 슬라이드에 모기를 주입하고 복구. 신선한 hemolymph의 희석제 12 μl를 데리러와 mesothorax의 측면 측면에서 모기에이 볼륨을 삽입하는 microinjector을 설정합니다. 주입되는 미네랄 오일을 방지하기 위해만큼 - 희석제의가 아닌 전체 볼륨 (10 μl 약 8) 가장 전달하지만. 희석액의 작은 볼륨 바늘의 끝에 유지됩니다. </li><li> 절단 복부 자리가 1cm의 원형의 가장자리에 있습니다 있도록 모기를 놓습니다. 주사 후 희석 hemolymph가 표시된 지역 수집해야합니다. 바늘이 mesothoracic 주사 사이트에서 제거되면, 포셉의 두 팔 사이에 모기를 들고 다시 부드럽게 에칭 서클 지역에 hemolymph 직접 이러한 포셉와 복부를 당겨요. </li><li> 희석 hemolymph은 10 분 정도하거나 가시 건조까지 슬라이드에 건조하도록 허용합니다. </li></ol> 5. Hemocyte 고정 및 염색법 <ol><li> 딥 S : HEMA3 7 (자료 참조)와 별도로 각 슬라이드를 얼룩때마다 일초에 대한 정착액 5 회에 라이드, 각 일초과, 마지막으로, 솔루션 II에서 수영을 슬라이드 3 번, 나는 각각의 일초 3 시간이 솔루션 Dipslide. DI 워터 슬라이드의 뒷면을 씻어. 슬라이드의 앞에는 씻어서해서는 안됩니다. 스테인드 조직을 포함하는 에칭 동그라미 밖 슬라이드의 전면을 가릴 건조. </li><li> 에칭 중심 주위를 장착 매체 (예 : Shur / 마운트 또는 Polymount)를 적용하고 그 위에 덮개 용지를 넣으십시오. 그래서 커버 슬립 (25 X 25mm)로 가볍게 눌러되는 커버 슬립 (스테인드 조직을 포함하는 에칭 동그라미 포함)이 적용되는 전체 영역에 장착 매체 확산. 슬라이드 3 시간 또는 밤새 최소 건조하실 수 있습니다. Hemocytes는 최적의 시각화 및 이미징에 대한 수집 다음과 같은 이주 내에 볼 수 있습니다. </li></ol> 6. 대표 결과 Aedes aegypti 성인 여성에서 수집한 고정 및 HEMA3 스테인드 hemocytes의 예 그림 1A - C에 표시됩니다. 그림 1A 우리가 세포질 비율 (550x 배율) 11 핵 작은 크기, 구형 - 회전 타원체의 형태와 높이에 따라 prohemocyte로 식별되는 hemocyte를 보여줍니다. 그림 1B는 핵 비율 (550x 배율)하​​기 위해 회전 타원체의 형상과 높은 세포질에 따라 oenocytoid로 분류 hemocyte를 보여줍니다. 마지막으로, 그림 1C는 과립 타입 hemocyte (550x 배율)를 보여줍니다. Granulocytes 자연에 더 세분화하고 그들이 유리 표면을 준수 같은 모양과 행동에 더 amoeboid 경향이 있습니다. hemocyte 입력 및 복구 11 이전에 게시된 기준을 사용하여, 우리의 수집 방법은 총 hemocytes의 비슷한 번호를 굴복 우리는 마찬가지로 granulocytes 총 hemocytes의 가장 큰 비율 11 (그림 2) 메이크업 것으로 나타났습니다. <img src="/files/ftp_upload/2772/2772fig1.jpg" alt="그림 1" /> 그림 1. 대표 HEMA3 고정, 스테인드 prohemocyte의 빛 현미경 이미지 (A), oenocytoid (B), 그리고 애의 과립 (C). aegypti 성인 여성. C = 세포질, 핵 N = G = 과립. <img src="/files/ftp_upload/2772/2772fig2.jpg" alt="그림 2"/> 그림 2. 총 카운트 ± 저희 hemolymph 희석 및 수집 방법으로 여성 모기 당 얻은 모든 hemocytes와 granulocytes의 SE.

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관심 없음 충돌 선언하지 않습니다.

hemocyte 수집 방법은 이전에 게시 방법에서 수정 한 적은 수의 단계를 Aedes 모기에서 hemocytes의 깨끗한 준비를 분리 수있다 여기에서 설명한. 우리의 특별한 관심 Aedes 모기에 hemocyte 인구 특성화에 자리잡고 있지만, 우리는 초기 실험이 적절한 사출 볼륨을 결정하기 위해 실시 후이 기법은 다른 모기 그룹에 적용할 수 믿습니다. 우리 프로토콜은 생리 식염수 대신 항응고제 버퍼를 사용?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 슈나이더의 중간 </td><td> 시그마 - 알드리치 </td><td> S0146 </td><td></td></tr><tr><td> 태아 소 혈청 </td><td> 시그마 - 알드리치 </td><td> F0643 </td><td></td></tr><tr><td> Hema3 얼룩 키트 </td><td> 어부 </td><td> 123-869 </td><td></td></tr><tr><td> 유리 바늘 (필라멘트와 borosilicate) </td><td> 서터 악기 </td><td> BF100 - 78-10 </td><td> 1.0mm OD 및 0.78mm ID </td></tr><tr><td> 니들 풀러 </td><td> 서터 인 스트 루먼트 </td><td> 모델 P - 87 </td><td></td></tr><tr><td> MicroInjector </td><td> Tritech 연구 </td><td> MINJ - PD </td><td></td></tr><tr><td> 니들 홀더 </td><td> Tritech 연구 </td><td> MINJ - 4 </td><td></td></tr></tbody></table>

a protocol for collecting and staining hemocytes from the yellow fever mosquito aedes aegypti

모기는 황열병 바이러스, 말라리아 기생충 및 filarial 웜과 같은 질병을 유발 병원균의 번호를 벡터입니다. 실험실은 병원균 1, 2 내화물 아르 유전자 변형 모기를 생성하는 희망에서 질병 벡터 수종에 본래 면역 시스템의 백신 병원체 구성 요소를 조사하고 있습니다. 모기의 타고난 면역 시스템은 방어 3 몇 가지 라인으로 구성되어 있습니다. 상피 - 줄지어 모기 midgut 사에 의해 부과된 장벽을 탈출하기 위해 관리 병원균은 hemolymph를 입력 순환 hemocytes, 캡슐 중요한 세포 구성 요소와 삼켜의 병원균 5, 6을 발생합니다. 연구팀은 모기의 조혈 조직에 대한 증거를 발견하지 않은 현재의 증거는 hemocytes의 숫자가 성인 출현에 고정하고 숫자가 실제로 모기 세 7로 거부할 수있는 제안합니다. 제대로 수집하고 의료 중요한 곤충의 hemocytes를 식별하는 능력은 세포 면역의 연구에 필수적인 단계입니다. 그러나, 모기의 작은 크기와 hemolymph의 제한된 볼륨 면역 세포를 수집하는 도전을 제기. 모기 hemocytes를 수집하기 위해 두 설립 방법은 상처 코 8 hemolymph의 추방, 그리고 호수가 머리와 흉부 (즉, 자궁 경부)와 perfused hemolymph가 수집되는 사이 막의 necklike 영역으로 주입되는 볼륨 변위 (재관류)를 포함 복부 9, 10의 말초 지역의 찢어진 오프닝에서. 이러한 기술은, 그러나, hemocytes의 낮은 복구 및 각각 11 지방 신체 세포에 의해 오염 가능성에 의해 제한됩니다. 오염 저울 및 내부 조직 11 레벨을 감소하면서 최근에는 방법이 항응고제 버퍼의 사용에 의해 immunocytes 높은 주입 / 복구 개선 복구이라고도합니다. 그 방법이 기본 문화 hemocytes를 수집 및 유지 관리의 개선 방법에 대한 수 있지만, 그것은 주사와 하류 목표가 진단에 대한 hemocytes를 수집, 수정하고 얼룩이있다면 필요하지 않습니다 수집 단계의 숫자를 알아볼 수 있습니다. 여기, 우리는 Aedes 모기에 hemocytes의 깨끗한 준비를 분리 높은 사출 기술의 정확성과 생리 식염수 대신에 항응고제 버퍼를 사용하여 재관류의 단순 결합 모기 hemolymph를 수집하는 우리의 방법을 보여줍니다.

Watch this Scientific Journal Video about A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti at JoVE.com

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A는 설명 모기 hemocytes를 수집하고 얼룩 아직 정확한 방법을 단순화. 우리의 방법은 Aedes 모기에 hemocytes의 깨끗한 준비를 분리 높은 사출 기술의 정확성과 재관류의 단순 결합. 이 방법은 연구가 필요한 hemocytes의 유형에 대한 지식과 풍부한을 용이하게합니다.

옐로 피버 모스키토에서 Hemocytes를 수집 및 스테 이닝을위한 프로토콜 Aedes aegypti</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

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JoVE Journal

Immunology and Infection

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

15.2K 조회수.  University of Richmond. A는 설명 모기 hemocytes를 수집하고 얼룩 아직 정확한 방법을 단순화. 우리의 방법은 Aedes 모기에 hemocytes의 깨끗한 준비를 분리 높은 사출 기술의 정확성과 재관류의 단순 결합. 이 방법은 연구가 필요한 hemocytes의 유형에 대한 지식과 풍부한을 용이하게합니다.

비디오: A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

쥐 말라리아 기생충의 유전자 크로스의 생산을위한 프로토콜

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

연구

면역학 및 감염병학

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes. The majority of them belong to the superfamily of G protein-coupled receptors (GPCRs). We have focused on characterizing arthropod kinin receptors from the tick and mosquito. Arthropod kinins are multifunctional neuropeptides with myotropic, diuretic, and neurotransmitter function. Here, a method for systematic analyses of structure-activity relationships of insect kinins on two heterologous kinin receptor-expressing systems is described. We provide important information relevant to the development of biostable kinin analogs with the potential to disrupt the diuretic, myotropic, and/or digestive processes in ticks and mosquitoes.
The kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the mosquito Aedes aegypti (Linnaeus), were stably expressed in the mammalian cell line CHO-K1. Functional analyses of these receptors were completed using a calcium bioluminescence plate assay that measures intracellular bioluminescence to determine cytoplasmic calcium levels upon peptide application to these recombinant cells. This method takes advantage of the aequorin protein, a photoprotein isolated from luminescent jellyfish. We transiently transfected the aequorin plasmid (mtAEQ/pcDNA1) in cell lines that stably expressed the kinin receptors. These cells were then treated with the cofactor coelenterazine, which complexes with intracellular aequorin. This bond breaks in the presence of calcium, emitting luminescence levels indicative of the calcium concentration. As the kinin receptor signals through the release of intracellular calcium, the intensity of the signal is related to the potency of the peptide.
This protocol is a synthesis of several previously described protocols with modifications; it presents step-by-step instructions for the stable expression of GPCRs in a mammalian cell line through functional plate assays (Staubly et al., 2002 and Stables et al., 1997). Using this methodology, we were able to establish stable cell lines expressing the mosquito and the tick kinin receptors, compare the potency of three mosquito kinins, identify critical amino acid positions for the ligand-receptor interaction, and perform semi-throughput screening of a peptide library. Because insect kinins are susceptible to fast enzymatic degradation by endogenous peptidases, they are severely limited in use as tools for pest control or endocrinological studies. Therefore, we also tested kinin analogs containing amino isobutyric acid (Aib) to enhance their potency and biostability. This peptidase-resistant analog represents an important lead in the development of biostable insect kinin analogs and may aid in the development of neuropeptide-based arthropod control strategies.

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

This protocol provides instructions for clonal-cell line selection and a calcium bioluminescence assay to analyze the structure-activity relationships of synthesized arthropod neuropeptides on their cognate GPCRs. This assay can be used for receptor deorphanization and structure-activity relationship studies for synthetic analog design and peptide/drug-lead discovery.

모기의 기능 분석을 위해 칼슘 Bioluminescence 분석 ( Aedes aegypti)와 틱 ( Rhipicephalus microplus) G 단백질 결합 수용체

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

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 ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

C 형 간염 바이러스의 복제를 분석하기위한 프로토콜

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

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 ...

Co-staining Blood Vessels and Nerve Fibers in Adipose Tissue

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of obesity. Therefore, developing an easy and efficient method to document the dynamic changes in adipose tissue is necessary. Here, we describe a modified immunofluorescent approach that efficiently co-stains blood vessels and nerve fibers in adipose tissues. Compared to traditional and recently developed methods, our approach is relatively easy to follow and more efficient in labeling the blood vessels and nerve fibers with higher densities and less background. Moreover, the higher resolution of the images further allows us to accurately measure the area of the vessels, amount of branching, and length of the fibers by open source software. As a demonstration using our method, we show that brown adipose tissue (BAT) contains higher amounts of blood vessels and nerve fibers compared to white adipose tissue (WAT). We further find that among the WATs, subcutaneous WAT (sWAT) has more blood vessels and nerve fibers compared to epididymal WAT (eWAT). Our method thus provides a useful tool for investigating adipose tissue remodeling.

New blood vessel formation and sympathetic innervation play pivotal roles in adipose tissue remodeling. However, there remain technical issues in visualizing and quantitatively measuring adipose tissue. Here we present a protocol to successfully label and quantitatively compare the densities of blood vessels and nerve fibers in different adipose tissues.

혈관과 신경 섬유 지방 조직에서 공동 얼룩

Recent studies have highlighted the critical role of angiogenesis and sympathetic innervation in adipose tissue remodeling during the development of ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

광견병 백신 효능을 평가하기 위한 시험관 내 ELISA 테스트

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

지카 바이러스를 사용하여 Aedes aegypti 모기에 벡터 역량 분석

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Aedes aegypti 모기에서 현미경 포리디안 기생충 에드hazardia aedis의 전파

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In the laboratory, researchers can deliver animal-derived and artificial blood meals to mosquitoes via membrane feeders, which allow for manipulation of meal composition. Here, we present methods for feeding blood and artificial blood meals to Aedes aegypti mosquitoes and quantifying the volume consumed by individual females.
Targeted feeding and quantification of artificial/blood meals have broad uses, including testing the effects of meal components on mosquito behavior and physiology, delivering pharmacological compounds without injection, and infecting mosquitoes with specific pathogens. Adding fluorescein dye to the meal prior to feeding allows for subsequent meal size quantification. The meal volume consumed by mosquitoes can be measured either by weight, if the females are to be used later for behavioral experiments, or by homogenizing individual females in 96-well plates and measuring fluorescence levels using a plate reader as an endpoint assay. Meal size quantification can be used to determine whether changing the meal components alters the meal volume ingested or if meal consumption differs between mosquito strains. Precise meal size quantification is also critical for downstream assays, such as those measuring effects on host attraction or fecundity. The methods presented here can be further adapted to track meal digestion over the course of days or to include multiple distinguishable markers added to different meals (like nectar and blood) to quantify the consumption of each meal by a single mosquito.
These methods allow researchers to singlehandedly perform high-throughput measurements to compare the meal volume consumed by hundreds of individual mosquitoes. These tools will therefore be broadly useful to the community of mosquito researchers for answering diverse biological questions.

Feeding and Quantifying Animal-Derived Blood and Artificial Meals in Aedes aegypti Mosquitoes

The goal of this protocol is to deliver animal-derived and artificial blood meals to Aedes aegypti mosquitoes through an artificial membrane feeder and precisely quantify the volume of meal ingested.

Aedes aegypti 모기에서 동물 유래 혈액 및 인공 식사 먹이주기 및 정량화

Females of certain mosquito species can spread diseases while biting vertebrate hosts to obtain protein-rich blood meals required for egg development. In ...