이 문서에 보고된 사업은 국민 건강과 호주 프로그램 그랜트 (CRP)과 NHMRC 프로젝트 그랜트 (CRP 및 BJCQ)의 의학 연구 협의회 (NHMRC)에 의해 지원되었다. 또한 BJCQ은 NHMRC 피터 도허티 박사 과정 이수 연구원입니다.

1) 시약 설정 1.1) CFSE 주식 솔루션 CFSE의 염료는 일반적으로 25 MG의 튜브에 carboxyfluorescein 디아 세 테이트 succinimidyl 에스테르 (CFDA, SE) (MW 557.47 g / 두더지)로 구입합니다. (몇 개월 -20 ° C에서 50-100 μL aliquots로 저장) 5 mm의 최종 주식 솔루션 8.96 ML DMSO에 25 밀리그램을 디졸브. 이 같은 노출은 저장하는 동안 피해야하는 것이 중요하므로 CFDA, SE는 수용액과 반​​응합니다. 1.2) Lymphocytes 마우스의 비장 및 또는 림프절에서 lymphocytes 격리하고 함께 문화 매체 1 ML (예 : RPMI)에 resuspend 5% 열 inactivated (HI) 사이 0.5 × 10 6 세포 농도와 태아 송아지 혈청 (FCS), - 10 X 10 7 / ML. 2) CFSE 라벨 2.1) 정기 방법 : <ol class="lalpha"><li> 완전히 새로운 (비 침수) 10 ML 원뿔 튜브의 바닥에 신중하게 중간과 장소의 하나 ML 볼륨에서 세포를 resuspend. </li><li> (비 침수 튜브를 사용하여 이동하고 성급하게 CFDA, SE 솔루션을 혼합에서 한 ML - 세포 현탁액을 방지합니다) 수평 튜브를 놓는다. </li><li> 조심스럽게 그것은 세포 솔루션과의 접촉을하지 않는 보장 튜브의 상단에있는 플라스틱 이외의 침수 부분에 PBS 110 μL를 추가합니다. </li><li> 110 μL PBS에 CFDA, SE의 5 MM 주식 1.1 μL를 Resuspend. </li><li> 신속 튜브 뚜껑 및 솔루션의 빠른 혼합 제복을 얻으려면 잘 반전하고 와동. CFSE의 염료 5 μm의의 최종 농도에됩니다 그러나, 최적 농도는 준비 각 배치에 대해 결정 할 수 있습니다, 그리고 그것이 효과가 있도록 6 개월마다 검사. </li><li> 상온에서 5 분 세포를 품어. CFSE에 CFDA, SE의 전환시 (토론 참조) 염료는 형광등과 과도한 빛에 노출에 의해 표백에 따라서 경향이된다고합니다. 그러므로, 그것은 알루미늄 호일로 덮고하여 예를 들어,이 시점에서 빛으로부터 튜브를 보호하는 것이 좋습니다. </li><li> 20 ° C와 폐기 뜨는에서 5 분 300 XG에서 원심 분리에 의해 sedimenting, 20 ° C PBS 5 % HI FCS를 포함 10 권 diluting하여 세포를 씻으십시오. 두 번 더 씻어 반복합니다. </li></ol> (10도 이상의 휴대 전화 번호에 적합한 2.2) 대체 방법 - 30 X 10 7) : <ol class="lalpha"><li> 1 PBS의 ML 신속하게 철저히 resuspended 세포 1 ML에 추가하기 위해 5 MM 주식 2 μL를 추가하여 2 X (10 μm의) CFDA, SE 솔루션을 준비하고 신속하게 튜브를 뚜껑과 반전 잘 소용돌이하려면 빠르고 균일 혼합. </li><li> 상온에서 5 분 세포를 품어 및 단계 2.1 (F) 및 2.1 (G)에서와 같이 씻으십시오. </li></ol> 2.3) 전지는 다음 세포 분열의 유도에 대한 관심이 프로토콜에 적용할 수 있습니다. 라벨 세포는 체외에서 생체내의 assays에서 모두 사용할 수 있습니다. 2.4) 확산 분석의 완성에 세포가 수확되고 (대표 예제 아래 참조) 유동세포계측법으로 분석했다. 3) 대표 결과 CFSE 희석하여 림프구 증식을 평가하기 위해서는 본격적 세포 (즉, 최대 형광)이 아닌 표시된 셀 (예 : 최소 형광)의 형광을 알고 유용합니다. 따라서 귀하의 실험 설계는 계정에 이러한 컨트롤을해야합니다. 다음은 체외에서 유동세포계측법에 의해 CD8 + T 세포의 분열을 측정하는 CFSE를 사용하여 생체내 assays에의 예입니다. 시험 관내 자극에서 3.1) 그림 1의 CFSE 프로필을 보여줍니다 CFSE - 라벨 ovalbumin (OVA) - 구체적인 T 세포 수용체 유전자 변형 OVA의 다양한 양의와 펄스 돌기 세포 3 일 문화 이후 CD8 + OT - I T 세포. CD8 + OT - I 생쥐의 비장과 림프 노드에서 정화 T 세포가 CFSE 및 3.3 X 10 4 DC와 교양 1 X 10 5 세포로 분류되었습니다. DC 어디 3​​7 1 시간에 대한 OVA ° C와 펄스와 문화 두 번 씻어 사전. 후 3 일 수확 및 레이블 방지 CD8 - APC 항체와 Hoechst 33258와 다음 유동세포계측법 (50000 이벤트가 수집)에 의해 분석 셀. 라이브는 (Hoechst 33258 -) CD8 + 세포가 표시됩니다. 비 표시된 세포가 자동 형광 세포가 감지 세포 분열의 한계를 보여줍니다 동안의 컨트롤로, 혼자 교양 CD8 + T 세포는, 비 나누어 세포의 CFSE 강도를 보여줍니다. 4 CFSE의 봉우리가 셀 3 부문까지받은 것을 나타내는이 예제에서 패널의 각 볼 수 있습니다. 생체내 자극에서 3.2) 그림 2는 생체내 3 일 후에 유전자 변형 CD8 + OT - I T 세포 CFSE - 분류 OVA 특정 T 세포 수용체의 CFSE 프로필을 보여줍니다 IN 20 μg의 OVA의 존재. CD8 + CD45.1 congenic OT - I 생쥐의 비장과 림프 노드에서 정화 T 세포가 CFSE 5 X 10 6 세포 C56BL/6J의 측면 꼬리 정맥 (CD45.2 +) 마우스로 IV를 주입으로 표시되었습니다. 2 시간 후 마우스 OVA 20 μg과 IV 주입했다. 삼일 후, 생쥐의 spleens는 안티 - B220 - PerCP, 안티 - CD8 - APC - Cy7 및 안티 CD45.1 - PE - Cy7 항체와 Hoechst 33258로 표시된, 단일 세포 현탁액으로 만든 수집하고 분석했다 유동세포계측법에 의해 (1,000,000 이벤트가 수집). 라이브 (Hoechst 33258는 -) B220 - 세포가 오른쪽 패널에 표시되는 왼쪽 패널과 문이 CD8 + CD45.1 + 세포에 표시됩니다. 컨트롤로, unstimulated CD8 CFSE - 표시 + T 세포가 아닌 나누어 세포의 CFSE 강도를 보여줍니다, 이외의 레이블이 붙은 세포가 세포가 감지 세포 분열의 한계의 자동 형광을 보여줍니다하는 동안. CD45 allotypic 차이의 사용이 전송 CD8를 해결 + 호스트에서 T 세포를, 그들은 전체 CD8 + T 세포 (왼쪽 패널)의 사소한 일부 아르로 매우 중요합니다. 대부분의 세포는 세포가 6 부문까지받은 것을 나타내는 예제 (오른쪽 패널) 7 CFSE의 봉우리 이내에 가을 있습니다. <img src="/files/ftp_upload/2259/2259fig1.jpg" alt="그림 1" /> 그림 1. CFSE - 라벨 ovalbumin (OVA) - 구체적인 T 세포 수용체 유전자 변형 CD8 OVA의 다양한 농도와 펄스 DC로 체외에 대응 + OT - I T 세포, 삼일 문화 뒤에되고 그림 데이터. 참고 CD8가 아닌 나누어 인구 + T 능력 항원 (밝은 회색의 히스토그램)와 비 표시된 인구 (어두운 회색 히스토그램)의 자동 형광의 부재에서 세포. 그림 CD8을 모습 + 높은 항원 농도에 나누어 더 많은 T 세포와 CFSE 희석 봉우리에 따라 1-3 회 분할이 T 세포. <img src="/files/ftp_upload/2259/2259fig2.jpg" alt="그림 2" /> 그림 2. 능력 CFSE - 라벨 ovalbumin (OVA) - 특정 adoptively OVA에 대응하는 C57BL / 6 마우스에 양도 T 세포 수용체 유전자 변형 CD8 + OT - I T 세포, 입양이 완료된 후 삼일 표시된 데이터는 왼쪽 패널 :. CD8 +, CD45. 받는 마우스 1 + OT - 1 T 세포. 오른쪽 패널 : CFSE 확산 프로필. CD8가 아닌 분할 인구 참고 +받는 생쥐의 T 세포는 항원 (밝은 회색의 히스토그램)와 비 표시된 lymphocytes (어두운 회색 히스토그램)의 자동 형광을받지 않습니다. 그림 CFSE 희석 봉우리에 따라 1-6 번 나누어 가지고 CD8 + T 세포를 묘사. <img src="/files/ftp_upload/2259/2259fig3.jpg" alt="그림 3" /> 그림 3. carboxyfluorescein 디아 세 테이트 succinimidyl 에스테르 (CFDA, SE)와 함께 세포의 라벨 중에 발생하는 여러 가지 분자 이벤트의 표현. 프로세스에 대한 자세한 내용은 토론 텍스트를 참조하십시오. 참고 : 일반적인 약어 &#39;CFSE&#39;가 아닌 CFDA, SE는 일반적으로 라벨 시약 및 라벨 절차를 설명하는 데 사용됩니다. 파리쉬 4 기반의 그림.

<ol>
	<li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171, 131-137 (1994).</li><li>Parish, C. R., Glidden, M. H., Quah, B. J., Warren, H. S., Coligan, 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=Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation.">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current protocols in immunology. , (2009).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nature Protocols. 2, 2049-2056 (2007).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77, 499-508 (1999).</li></ol>

관심 없음 충돌 선언하지 않습니다.

CFSE 작동 왜 빠른 균일한 라벨이 확산 분석의 사용하기 위해 필요한 것이 얼마나 감사하기 위해, 그것은 CFSE 셀 라벨의 분자 기초를 이해하는 데 유용합니다. 이것은 두 단계로, 1) 셀 항목과 2) 단백질 라벨 (그림 3)으로 나눌 수 있습니다. 1) 셀 입력 : 세포에 염색의 항목은 염료의 diacetylated 양식 carboxyfluorescein 디아 세 테이트 succinimidyl 에스테르 (CFDA, SE)에 의해 중재됩니다. acetates는 플라즈마 막 전체의 빠른 흐름을 허용 염료 높은 막 permeant합니다. 세포 내에서 현재 Esterases, 따라서 클리브 CFDA, SE에서 acetates 그리고 훨씬 더 적은 막 permeant있는 염료의 CFSE 양식을 일으키다가, 따라서 세포 내에 염료를 집중. 2) 단백질 라벨 : CFDA, SE 및 CFSE 모두 아미노 반응 succinimidyl 사이드 체인을 가지고 있지만, 오직 CFSE는 형광등입니다. CFSE의 높은 세포 농도는 단백질의 신속하고 높은 수준의 세포 라벨을 용이하게합니다. 세포의 CFSE 라벨은 대표적인 결과를 (그림 1 및 2)와 같이 여러 부서를받은 세포를 해결하기위한 중요 세포의 homogenously 라벨 인구를 얻기 위해 신속하게 수행되어야합니다.

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		<thead>
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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>CFDA,SE</td>
<td>&nbsp;</td>
<td>Molecular Probes</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DMSO</td>
<td>&nbsp;</td>
<td>Cambridge Isotope Laboratories Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lymphocytes</td>
<td>&nbsp;</td>
<td>eg mouse or human</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>SAFC Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10-15ml conical tubes</td>
<td>&nbsp;</td>
<td>Falcon, Becton Dickinson</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Aluminium foil</td>
<td>&nbsp;</td>
<td>Capral Aluminium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vortex</td>
<td>&nbsp;</td>
<td>Scientific Industries</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>&nbsp;</td>
<td>Eppendorf (5810 R)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>LSR-II (BD Biosciences)</td>
<td>&nbsp;</td>
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the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

Carboxyfluorescein succinimidyl 에스테르 (CFSE)는 림프구 분열 1-3를 모니터할 수있는 효과적이고 인기있는 수단입니다. CFSE는 covalently 형광 염료, carboxyfluorescein와 긴 세포내 분자를 표시합니다. 따라서, CFSE - 표시 셀 분할 때, 그 자손은 carboxyfluorescein - 태그 분자의 절반 숫자 둘러 싸인하고 있으므로 각각의 세포 분열은 유동세포계측법를 통해 셀 형광에 해당 감소를 측정하여 평가하실 수 있습니다. 의 낮은 세포 독성과 결합된 매우 낮은 분산 높은 형광 강도와 림프구 인구, 라벨 CFSE의 용량은, 그 세포 분열을 측정하는 이상적인 염료합니다. 그것이 플루오레신 기반 염색이기 때문에 그것은 또한 멀티 컬러 유동세포계측법하는 것이 해당하는 다른 fluorochromes 광범와 호환됩니다. 이 문서는 일반적으로 8 셀 부문까지 감시하기위한 목적으로 마우스 lymphocytes를 라벨에 사용되는 절차를 설명합니다. 이 분류 세포는 체외에서 생체내 연구에 대해 모두 사용할 수 있습니다.

Watch this Scientific Journal Video about The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation at JoVE.com

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE는 covalently 형광 염료, carboxyfluorescein와 긴 세포내 분자를 표시합니다. 따라서, CFSE - 표시 셀 분할 때, 그 자손 이는 세포 분열을 평가하는 데 사용할 수있는 형광의 절반 금액을 보유하고 있습니다. 이 문서는 일반적으로 CFSE와 마우스 lymphocytes를 라벨에 사용되는 절차를 설명합니다.

림프구 확산을 모니터 Carboxyfluorescein 디아 세 테이트 Succinimidyl 에스터 (CFSE)의 사용

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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Research

JoVE Journal

Immunology and Infection

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

91.3K 조회수.  John Curtin School of Medical Research, Australian National University. CFSE는 covalently 형광 염료, carboxyfluorescein와 긴 세포내 분자를 표시합니다. 따라서, CFSE - 표시 셀 분할 때, 그 자손 이는 세포 분열을 평가하는 데 사용할 수있는 형광의 절반 금액을 보유하고 있습니다. 이 문서는 일반적으로 CFSE와 마우스 lymphocytes를 라벨에 사용되는 절차를 설명합니다.

비디오: The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

운동 유발성 Apoptosis 림프구의 결정을위한 손가락 - 스틱 혈액 샘플링 방법론

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

연구

면역학 및 감염병학

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS로 인한 세균 경쟁을 모니터링 할 비주얼 분석

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

사용<em&gt; 생체 내</em&gt; 이미징 신생아의 실험 쥐 거대 세포 바이러스 감염의 진행 상황을 모니터링 할 수

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

형광 단백질을 사용하면 아프리카 Trypanosome에서 Glycosome 역학을 모니터링하려면

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

C 형 간염 바이러스에 대한 인간 락토페린의 세포 흡수 및 관련 바이러스 활동을 모니터링하는 면역 형광

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

The Use of Flow Cytometry to Assess the State of Chromatin in T Cells

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in na&#239;ve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI

Flow cytometry can be utilized to assess the state of chromatin within T cells. This protocol allows scientists to interpret evidence of chromatin decondensation during T cell activation demonstrated by an increase in the mean florescence intensity (MFI) of fluorescent Histone H3 antibodies

유동 세포 계측법의 사용은 T 세포의 염색질의 상태를 평가하기 위해

During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

T 림프구 - 접착 분자 상호 작용의 연구에 대한 접착에서 전단 응력의 분석

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

검출<em&gt; 트리파노소마 (Trypanosoma) brucei</em자기 활성 셀 정렬 및 유동 세포 계측법에 의해&gt; 변형 표면 당 단백질 전환

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

B 형 간염 바이러스 리포터 시스템 개발은 복제주기의 초기 단계를 모니터링하기

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran Murine 및 인간의 기본 NK 세포의 Lentiviral 변환 효율 향상

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...