我々は、資金助成R01-AI026328のためのNIH（KAK）およびR01-AI079004（KAK）に感謝。

1。生殖器の除去<ol><li>承認されたガイドラインを使用してマウスを安楽死させる。低正中切開を行い、皮膚を撤回。 </li><li>卵管から膣口に、全体の生殖管を分離し、削除します。 </li><li>ハサミで縦管全体を開きます。 </li><li>完全RPMI-10/EDTAとペトリ皿の中で細かい部分（&lt;約1mm）（下式参照）に微細手術用のハサミを持つ生殖器を切り取り、に設定マグネチックスターラー上で室温で125mlのフラスコ内の組織をかき混ぜる15分。この操作を2回繰り返し、その後上皮および他の残骸を含む上清は、破棄されます。 </li><li>セルストレーナー（40μmの）を通してフラスコの内容物を注ぐ。完全培地でのEDTAの残留物を洗い流してください。 熱不活化したRPMI 500ミリリットル（Gibco）を、10％ウシ胎児血清（FBS）、5 mlのペニシリン/ストレプトマイシン、5mlの1M HEPES：完全RPMI/10。 完全RPMI-10/EDTA：10％5μMEとしたRPMI（同上）DTA </li></ol> 2。生殖器の組織を消化<ol><li> 20ミリリットル新鮮RPMI-10/collagenase持つ新しいフラスコ（下のレシピを参照してください）​​に組織片を移し、精力的に組織をかき混ぜるように設定マグネチックスターラーで1時間37℃で組織を消化。 完全RPMI-10/collagenase：使用前右、再構成するコラゲナーゼタイプVIII（σ; -20℃で保存するか、製造元の指示に従って）、450-500U/mlの最終濃度に完全RPMI/10に追加します。 </li><li>第一消化した後、氷上で細胞を維持し、100μmのセルストレイナーで上清を収集します。 </li><li>新しいフラスコに未消化の組織を移し、3つの別々の消化の合計のため新鮮な完全RPMI-10/collagenaseを使用して、ステップ2.1をさらに2回繰り返す。この時点で、目に見える組織片は、溶液中でないこと。 </li><li>プールのすべてのサンプルから一緒に清と100μm細胞ストレーナーを通して収集しています。 cをスピンダウンells。細胞ペレットをパーコール勾配を用いて分離されます。 </li></ol> 3。パーコール勾配を用いて生殖管細胞の分離<ol><li>使用前パーコールの各濃度を準備します。 <ul><li> 100パーセントアイソトニックパーコール：ミックス9時01株式パーコール（ファルマシアBiotch）対10倍のPBS。 HClを用いてpHを7.4にそれを。 </li><li> 40パーセントPercol液：100％等張パーコールと完全RPMI 60mlの混合40ミリリットル。 </li><li> 70％パーコール液：完全RPMI 30mlでミックス100パーセントアイソトニックパーコールを70ml。 </li></ul></li><li> 40％パーコールソリューションのステップ6から各細胞ペレットを再懸濁します。 70％パーコール等量の勾配を作るために使用されます。勾配管の設定には2通りの方法があります。 <ol><li>層の下で：慎重に下地層70％パーコールその後、最初の勾配管に40％パーコールと細胞懸濁液を追加します。 </li><li>上層：最初のチューブに70％パーコールを追加して、慎重にゆっくりと上のセルを含む40％パーコールを読み込むトップ。 </li></ol></li><li>グラデーションのサンプルは、ブレーキをオフにして20分間、@ 900×gで、室温で遠心分離されます。 </li><li>リンパ球は40％と70％パーコール層の間の界面層になります。慎重に、界面層を採取したRPMI午前1時03分で洗い、740 xgでスピンダウンします。リンパ球は、細胞ペレットにされ、さらに使用するために準備ができているでしょう。 </li></ol> 4。代表的な結果マウス生殖管及びフローサイトメトリー（FACS）分析からのリンパ球の分離の例を図1に示します。生殖器は7日、感染後膣内感染マウスクラミジアmuridarumから削除されました。二つの生殖器管をFACSによって、機能と表現型検査のための十分なリンパ球を持たせるために、一緒にプールした。上記で概説したように解剖した組織は、消化や離工程で処理された。単一細胞懸濁液は、様々な蛍光標識染色したマウスCD3、CD4およびCD8に対するモノクローナル抗体。図1は、CD3陽性Tリンパ球にゲートをCD8陽性T細胞に対するCD4陽性T細胞のドットプロットの表示を示しています。私たちの手順は、マウスモデルで異なる疾患で様々な免疫細胞の機能と表現型の特徴を評価するために、異なった疾患モデルの生殖器官から単離したリンパ球を調べることができます。これらには、樹状細胞などの多様な免疫細胞は、好中球、マクロファージ、NK細胞、NKT細胞、T細胞（抗原特異的T細胞）、B細胞等3月10日が含まれています。 <img alt="図1" src="/files/ftp_upload/4391/4391fig1.jpg" /> 図1：リンパ球はここに示す方法を用いて、 クラミジアmuridarumに感染したマウスの生殖器官から単離した。完全分離手順の後、リンパ球を蛍光標識、CD3、CD4およびCD8で染色した。象限データは、CD3 +リンパ球にゲートをかけたCD4陽性を示す+とCD8 +とし、ゲート細胞の割合。

<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>

特別な利害関係は宣言されません。

このプロトコルでは、マウスの生殖器管からのリンパ球を準備する方法を示されており、容易に習得されています。このプロセスの最も重要な点は、氷の上に細胞を保持することによって、細胞の生存率を維持することです。このメソッドから細胞収率は、スキル、マウスの感染状況（ナイーブまたは感染翌日）、病原体（細菌、ウイルス、真菌）及びマウス生殖器官検討のセクション（卵?...

<table border="1"><tbody><tr><td> 名前 </td><td> 会社 </td><td> カタログ番号 </td><td></td></tr><tr><td>コラゲナーゼ</td><td>シグマライフサイエンス</td><td> C2139-1G </td><td></td></tr><tr><td>パーコール</td><td> GEヘルスケアバイオサイエンス</td><td> 17-0891-01 </td><td></td></tr><tr><td> RPMI1640 </td><td>ライフテクノロジーズギブコ</td><td> 11875 </td><td></td></tr></tbody></table>

isolation of lymphocytes from mouse genital tract mucosa

、消化器泌尿生殖器、及び気道では、このようなウイルスや細菌などの病原体1のエントリのポータルを提供するなど、粘膜表面。粘膜は、腸管内Peyersのパッチや気道における鼻関連のリンパ網内系組織などの病原体に対する免疫を生成するために、ホストの誘導サイトです。このユニークな機能は、生体防御システムの重要なプレーヤーとしての粘膜免疫をもたらします。多くの研究は、消化器、呼吸器の粘膜部位に焦点を当てている。しかし、生殖粘膜部位の少し調査があった。生殖器の粘膜は、細菌やウイルス感染などの性感染症（STD）のための一次感染のサイトです。性感染症は、今日の世界が直面する最も重要な健康課題の一つです。疾病管理予防センターは、米国で19万人が新たに感染毎年があると推定している。 STDsは毎年2米国の医療システムを170億ドルを費用、および即時、生涯健康への影響のコスト個人一層。この課題に立ち向かうためには、生殖粘膜免疫のより深い理解が必要とされ、リンパ球を分離し、これらの研究の重要なコンポーネントです。ここでは、再現性、他の種への適応のために変更することができる免疫学的研究のためのマウス雌の生殖器官からのリンパ球を単離するための方法を提示する。以下に説明する方法は、1つのマウスに基づいています。

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

マウス生殖管からリンパ球を分離するための効率的な方法が記載されている。この方法では、効率的な分離を可能にするために酵素消化し、パーコール勾配分離を利用しています。この手法は、他の種で使用するためにもに適応可能である

マウス生殖管粘膜からのリンパ球の単離

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 ビュー.  University of California, Los Angeles. マウス生殖管からリンパ球を分離するための効率的な方法が記載されている。この方法では、効率的な分離を可能にするために酵素消化し、パーコール勾配分離を利用しています。この手法は、他の種で使用するためにもに適応可能である

ビデオ: 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.

マウス尿路感染症の経尿道的誘導

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

免疫・感染学

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.

のRNAの単離緑膿菌マウス消化管に定着

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.

マウスのパイエル板のリンパ球や樹状細胞を単離し、免疫染色

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.

離とナイーブCD4 Tリンパ球のTh17分化

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-FISHによるマウスのBリンパ球における染色体異常の迅速分析

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.

ヒトパラティーノ扁桃腺からBおよびTリンパ球の効率的な単離プロトコール

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.

マウスの小腸リンパ球の単離およびフローサイトメトリーキャラクタリゼーション

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.

単離とマウスリンパ球の活性化

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.

Ex Vivoひと免疫不全ウイルス 1 における抗癌剤感受性との人間のリンパ組織と女性の性器粘膜の感染症

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.

人間の女性の生殖器官から樹状細胞の表現型および機能研究の分離

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