우리는 기금 보조금 R01-AI026328 (KAK) 및 R01-AI079004 (KAK)에 대한 NIH에 감사드립니다.

1. 성기 기관의 제거 <ol><li> 승인 된 지침을 사용하여 마우스를 안락사시켜야. 낮은 중간 선을 절개하고 피부를 철회. </li><li> 수란관에서 질구까지 전체 성기 기관을 분리하고 제거합니다. </li><li> 가위로 길이 방향 전체 기관을 엽니 다. </li><li> 전체 RPMI-10/EDTA있는 배양 접시에서 훌륭한 조각 (&lt;1mm 정도) (아래의 공식을 참조)로 미세 수술 가위로 성기 기관을 잘라로 설정 자석 교반기에 실온에서 125 ML 플라스크에 조직을 저어 15 분. 이 절차는 두 번 반복되는 다음 상피와 다른 파편을 포함하는 표면에 뜨는는, 삭제됩니다. </li><li> 셀 스트레이너 (40μm)를 통해 플라스크의 내용을 넣어. 전체 매체 EDTA (에틸렌 다이아 민 테트라 초산)의 잔류 물을 씻어. 열 inactivated RPMI 500 ML (Gibco), 10 % 태아 소 혈청 (FBS),, 5 ML 페니실린 / 스트렙토 마이신, 5 ML 100 HEPES : 전체 RPMI/10. 전체 RPMI-10/EDTA : 5μM E와 10% RPMI (위 등)DTA </li></ol> 2. 성기 요로 조직을 소화 <ol><li> 20 ML 신선한 RPMI-10/collagenase로 새 플라스크 (아래의 제조법 참조) 조직 조각을 전송하고 적극적으로 조직을 저어하도록 설정 자석 교반기에 1 시간에 37 ° C에서 조직을 소화. 전체 RPMI-10/collagenase : 마우스 오른쪽 사용하기 전에, reconstitute collagenase를 입력 VIII이 (시그마, -20에 저장 ° C 또는 제조업체의 지시에 따라.), 450-500U/ml의 최종 농도에 완벽한 RPMI/10에 추가 할 수 있습니다. </li><li> 첫 번째 소화 한 후, 100 μm 셀 스트레이너를 통해 supernatants를 수집 얼음에 세포를 유지. </li><li> 새로운 플라스크에 소화되지 않은 조직을 전송, 3 별도의 digestions의 총 신선한 완전한 RPMI-10/collagenase를 사용하여 단계 2.1 두 번 반복합니다. 이 시점에서 눈에 띄는 조직 조각이 솔루션에 할 수 없습니다. </li><li> 수영장 모든 샘플에서 함께 supernatants 100 μm 셀 스트레이너를 통해 수집합니다. C를 스핀아래 ells. 세포 펠릿은 Percoll의 그라디언트를 사용하여 구분됩니다. </li></ol> 3. Percoll 그라디언트를 사용하여 성기 요로 세포를 분리 <ol><li> 사용하기 전에 Percoll의 각 농도 준비 : <ul><li> 백퍼센트 등장 Percoll : 혼합 9시 1분 주식 Percoll (Pharmacia Biotch) 대 10 배 PBS. HCL을 사용하여 7.4으로 산도를. </li><li> 40 % Percoll 솔루션 : 혼합 백퍼센트 등장 Percoll과 완벽한 RPMI 60 ML 40 ML. </li><li> 70 % Percoll 솔루션 : 전체 RPMI의 30 ML과 혼합 백퍼센트 등장 Percoll의 70 ML. </li></ul></li><li> 40 % Percoll 솔루션의 6 단계에서 각 셀 펠렛을 Resuspend. 70 % Percoll의 동일한 볼륨은 그라디언트를 만들기 위해 사용됩니다. 그라데이션 튜브를 설정하는 방법은 두 가지가 있습니다 : <ol><li> 아래 층 : 그럼 조심스럽게 이하 계층 70 %의 Percoll 먼저 그라디언트 관에 40 % Percoll과 세포 현탁액을 추가합니다. </li><li> 이상 층 : 첫 번째 관에 70 % Percoll을 추가 한 다음 조심스럽게 천천히의 세포를 포함하는 40 % Percoll을로드맨. </li></ol></li><li> 그라디언트 샘플은 브레이크를 해제로 20 분을 위해, @ 900 XG, 방 온도를 centrifuged 될 것입니다. </li><li> 림프구는 40 %와 70 % Percoll 레이어 사이의 인터페이스 계층에있을 것입니다. 조심스럽게, 인터페이스 레이어를 수확 RPMI 1시 3분로 씻고 740 XG에서 스핀 다운. 림프구는 세포 펠렛에 추가로 사용할 준비가됩니다. </li></ol> 4. 대표 결과 마우스 성기 요로 및 유동 세포 계측법 (FACS) 분석에서 림프구의 분리의 예는 그림 1에 표시됩니다. 성기 요로 7 일 감염 후 intravaginally 감염 마우스 클라미디아 muridarum에서 제거되었습니다. 두 성기 책자는 FACS에 의해 기능 및 표현형 검사에 충분한 림프구를 위해 함께 풀링되었습니다. 해부 조직은 위에서 설명한대로 소화 및 절연 단계에서 처리되었다. 하나의 세포 현탁액은 다양한 fluorochrome - 복합에 얼룩이되었습니다마우스에는 3 번 CD, CD4 및 CD8에 대한 단클론 항체. 그림 1은 3 번 CD 긍정적 인 T 림프구의 출입을 금지 CD8 양성 T 세포 대 CD4 양성 T 세포의 점 플롯 프리젠 테이션을 보여줍니다. 우리 절차는 마우스 모델에서 다른 질병에 다양한 면역 세포 기능과 phenotypic 특성을 평가하기 위해 다른 질병 모델 성기 책자로부터 격리 림프구을 검사 할 수 있습니다. 이는 수지상 세포, 호중구, 대 식세포 NK, NKT, T 세포 (자세 별 T 세포), B 세포 등 3-10 등 다양한 면역 세포 등이 있습니다. <img alt="그림 1" src="/files/ftp_upload/4391/4391fig1.jpg" /> 그림 1. 림프구는 여기에 제시된 방법을 사용하여 클라미디아 muridarum에 감염된 생쥐의 성기 책자에서 고립되었다. 전체 분리 절차 후, 림프구는 fluorochrome - 복합 3 번 CD, CD4와 CD8 물들했다. 분면 데이터는 3 번 CD +의 림프구에 대한 출입을 금지했다CD4를 보여주는 + 대 CD8 +와와 출입을 금지 세포의 비율입니다.

<ol>
	<li>M, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+perinatal+immune+development+on+mucosal+homeostasis+and+chronic+inflammation.">The impact of perinatal immune development on mucosal homeostasis and chronic inflammation.</a> Nat. Rev Immunol. 12, 9-23 (2011).</li><li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2010 Sexually Transmitted Diseases Surveillance. , (2010).</li><li>Jain, S., Patrick, A. J., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+tandem+copies+of+conserved+gp41+epitopes+incorporated+in+gag+virus-like+particles+elicit+systemic+and+mucosal+antibodies+in+an+optimized+heterologous+vector+delivery+regimen.">Multiple tandem copies of conserved gp41 epitopes incorporated in gag virus-like particles elicit systemic and mucosal antibodies in an optimized heterologous vector delivery regimen.</a> Vaccine. 28, 7070-7080 (2010).</li><li>Tang, V. A., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravaginal+infection+with+herpes+simplex+virus+type-2+(HSV-2)+generates+a+functional+effector+memory+T+cell+population+that+persists+in+the+murine+genital+tract.">Intravaginal infection with herpes simplex virus type-2 (HSV-2) generates a functional effector memory T cell population that persists in the murine genital tract.</a> J. Reprod. Immunol. 87, 39-44 (2010).</li><li>Gillgrass, A. E., Tang, V. A., Towarnicki, K. M., Rosenthal, K. L., Kaushic, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+against+genital+herpes+infection+in+mice+immunized+under+different+hormonal+conditions+correlates+with+induction+of+vagina-associated+lymphoid+tissue.">Protection against genital herpes infection in mice immunized under different hormonal conditions correlates with induction of vagina-associated lymphoid tissue.</a> J. Virol. 79, 3117-3126 (2005).</li><li>Sajic, D., Patrick, A. J., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+delivery+of+CpG+oligodeoxynucleotides+expands+functional+dendritic+cells+and+macrophages+in+the+vagina.">Mucosal delivery of CpG oligodeoxynucleotides expands functional dendritic cells and macrophages in the vagina.</a> Immunology. 114, 213-224 (2005).</li><li>Jiang, J. Q., Patrick, A., Moss, R. B., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD8++T-cell-mediated+cross-clade+protection+in+the+genital+tract+following+intranasal+immunization+with+inactivated+human+immunodeficiency+virus+antigen+plus+CpG+oligodeoxynucleotides.">CD8+ T-cell-mediated cross-clade protection in the genital tract following intranasal immunization with inactivated human immunodeficiency virus antigen plus CpG oligodeoxynucleotides.</a> J. Virol. 79, 393-400 (2005).</li><li>Gill, N., Chenoweth, M. J., Verdu, E. F., Ashkar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NK+cells+require+type+I+IFN+receptor+for+antiviral+responses+during+genital+HSV-2+infection.">NK cells require type I IFN receptor for antiviral responses during genital HSV-2 infection.</a> Cell Immunol. 269, 29-37 (2011).</li><li>Thatte, A., DeWitte-Orr, S. J., Lichty, B., Mossman, K. L., Ashkar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+role+for+IL-15+in+TLR-mediated+innate+antiviral+immunity+against+genital+HSV-2+infection.">A critical role for IL-15 in TLR-mediated innate antiviral immunity against genital HSV-2 infection.</a> Immunol. Cell Biol. 89, 663-669 (2011).</li><li>Kwant-Mitchell, A., Ashkar, A. A., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+innate+and+adaptive+immune+responses+against+herpes+simplex+virus+type+2+in+a+humanized+mouse+model.">Mucosal innate and adaptive immune responses against herpes simplex virus type 2 in a humanized mouse model.</a> J. Virol. 83, 10664-10676 (2009).</li><li>Gallichan, W. S., Rosenthal, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+the+estrous+cycle+on+local+humoral+immune+responses+and+protection+of+intranasally+immunized+female+nice+against+herpes+simplex+virus+type+2+infection+in+the+genital+tract.">Effects of the estrous cycle on local humoral immune responses and protection of intranasally immunized female nice against herpes simplex virus type 2 infection in the genital tract.</a> Virology. , 224-487 (1996).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 프로토콜에서, 마우스 성기 책자에서 림프구를 준비하는 방법에 표시되며 쉽게 마스터 수 있습니다. 이 과정의 가장 중요한 포인트는 얼음에 세포를 유지하여 세포 생존 능력을 유지하는 것입니다. 이 방법에서 세포 수율은 기술, 마우스의 전염성 상태 (나이브 또는 감염 후 일), 병원체 (세균, 바이러스, 곰팡이)와 손쥐 생식 기관 심사의 절 (수란관, 자궁, 처리, 기술에 따라 달라집니다 질 세?...

<table border="1"><tbody><tr><td> 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td></td></tr><tr><td> Collagenase </td><td> 시그마 생명 과학 </td><td> C2139-1G </td><td></td></tr><tr><td> Percoll </td><td> GE 헬스 케어 바이오 과학 </td><td> 17-0891-01 </td><td></td></tr><tr><td> RPMI1640 </td><td> 생명 기술의 Gibco </td><td> 11,875 </td><td></td></tr></tbody></table>

isolation of lymphocytes from mouse genital tract mucosa

, 위장병 비뇨 생식 및 호흡기 책자에서, 바이러스 및 박테리아 1로 병원균에 대한 항목의 포털을 제공 등의 점막 표면. Mucosae는 창자에있는 Peyers 패치와 호흡기의 코 - 관련 lymphoreticular 조직 등의 병원균에 대한 면역을 생성하는 것도 호스트의 유도 사이트입니다. 이 독특한 기능은 호스트 방어 시스템의 중요한 플레이어로 점막 면역을 제공합니다. 많은 연구 위장병과 호흡기 점막 사이트에 초점을 맞추고있다. 그러나, 생식 점막 사이트의 약간의 조사가되었습니다. 성기 요로 점막은 세균과 바이러스 감염 등의 성병 (STD)의 기본 감염 사이트입니다. 성병은 오늘날 세계적가 직면하고있는 가장 중요한 건강 문제 중 하나입니다. 질병 통제 예방 센터는 미국 19 명의 새로운 전염성 매년있는 것으로 추정하고 있습니다. STDS 매년 2 미국 의료 시스템에서 1백70억달러 비용, 그리고 즉각적이고 평생 건강 결과에 비용 개인보다. 이 문제를 직면하기 위해, 생식 점막 면역의 큰 이해가 필요하고 림프구를 분리하면 이러한 연구의 필수 구성 요소입니다. 여기, 우리는 reproducibly 다른 종에 대한 적응을 위해 수정할 수 있습니다 면역 연구 손쥐 여성 성기 책자에서 림프구를 분리하는 방법을 제시한다. 아래에서 설명하는 방법은 한 마우스에 기반을두고 있습니다.

Watch this Scientific Journal Video about Isolation of Lymphocytes from Mouse Genital Tract Mucosa at JoVE.com

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

마우스 성기 기관에서 림프구를 분리 할 수​​있는 효과적인 방법이 설명되어 있습니다. 이 방법은 효율적으로 분리 할 수​​ 있도록 효소 분해와 Percoll 기울기 분리에 활용합니다. 이 기술은 다른 종에 사용하기 위해도에 적용 할 수 있습니다

마우스 성기 요로 점막에서 림프구의 분리

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

isolation-of-lymphocytes-from-mouse-genital-tract-mucosa

Research

JoVE Journal

Immunology and Infection

14.2K 조회수.  University of California, Los Angeles. 마우스 성기 기관에서 림프구를 분리 할 수​​있는 효과적인 방법이 설명되어 있습니다. 이 방법은 효율적으로 분리 할 수​​ 있도록 효소 분해와 Percoll 기울기 분리에 활용합니다. 이 기술은 다른 종에 사용하기 위해도에 적용 할 수 있습니다

비디오: Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Transurethral Induction of Mouse Urinary Tract Infection

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on Luria-Bertani (LB) agar at 37&deg;C in ambient air. UPEC strains grow as yellowish-white translucent colonies on LB agar. Following confirmation of appropriate colony morphology, single colonies are then picked to be cultured in broth. LB broth can be used for most uropathogenic bacterial strains. Two serial, overnight LB broth cultures can be employed to enhance expression of type I pili, a well-defined virulence factor for uropathogenic bacteria. Broth cultures are diluted to the desired concentration in phosphate buffered saline (PBS). Eight to 12 week old female mice are placed under isoflurane anesthesia and transurethrally inoculated with bacteria using polyethylene tubing-covered 30 gauge syringes. Typical inocula, which must be empirically determined for each bacterial/mouse strain combination, are 106 to 108 cfu per mouse in 10 to 50 microliters of PBS. After the desired infection period (one day to several weeks), urine samples and the bladder and both kidneys are harvested. Each organ is minced, placed in PBS, and homogenized in a Blue Bullet homogenizer. Urine and tissue homogenates are serially diluted in PBS and cultured on appropriate agar. The following day, colony forming units are counted.

This video will demonstrate methods to transurethrally induce mouse urinary tract infections and quantify the extent of resulting infections.

마우스 요로 감염의 Transurethral 유도

Uropathogenic bacterial strains of interest are grown on agar. Generally, uropathogenic E. coli (UPEC) and other strains can be grown overnight on ...

연구

면역학 및 감염병학

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

의 RNA 격리 녹농균 Murine 위장관을 Colonizing

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

림프구와 손쥐 Peyer의 패치에서 수지상 세포를 분리하고 Immunostaining

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&amp;#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

분리 및 나이브 CD4 T 림프구의 TH17 분화

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

PNA-FISH에 의해 마우스 B 림프구에서 염색체 이상을 신속하게 분석

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

인간 팔라티노 편도선에서 B와 T 림프구에 대한 효율적인 분리 프로토콜

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

쥐 소장 림프구의 분리 및 유세포 특성

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

분리 및 쥐 림프구의 활성화

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

전 비보 인간 면역 결핍 바이러스 1와 Histoculture 인간 림프 조직 및 여성 생식 기 점 막의 감염

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

Isolation of Dendritic Cells from the Human Female Reproductive Tract for Phenotypical and Functional Studies

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low numbers of DCs present per tissue. Yet, as the phenotype and function of DCs is modified by the tissue environment, it is necessary to analyze tissue resident DC populations, since blood derived DCs incompletely reflect the complexities of DCs in tissues. Here we present a protocol to isolate DCs from the human female reproductive tract (FRT) using hysterectomy specimens that allows both phenotypical and functional analyses. The protocol consists of tissue digestion to generate a single cell mixed cell suspension, followed by positive magnetic bead selection. Our tissue digestion protocol does not cleave surface markers, which allows phenotypical and functional analysis of DCs in the steady state, without overnight incubation or cell activation. This protocol can be adapted for the isolation of other immune cell types or isolation of DCs from other tissues.

We describe here a method to isolate and purify dendritic cells from different anatomical compartments in the human female reproductive tract for the evaluation of their phenotypical and functional characteristics. This method can be adapted to isolate other immune cells or dendritic cells from other mucosal tissues.

Phenotypical 및 기능 연구에 대 한 인간의 여성 생식 관에서 수지상 세포의 고립

The characterization of the human dendritic cells (DCs) resident in mucosal tissues is challenging due to the difficulty in obtaining samples, and the low ...