著者らは、マウスやウサギの彼らの手入れや、保守のために特別にベクトルを媒介とする疾病の動物資源支店の部門、アンドレアソン、リサマスウーディー、ヴァーナ·オブライエン、ジョン·リデルに感謝します。また、この原稿に向かって彼らの貢献のためにエイミー·ウルマン、テレサ·ラッセル、バーバラJ.ジョンソンに感謝します。最後に、この原稿の撮影に関連付けられているすべての合法性を導くためのグラフィックイラストやジュディ·ラヴェルを製造するためのCDCのコミュニケーションのためのアソシエートディレクターのオフィスでアリッサ·エッカートを確認したいと思います。

1。スライドの準備のために体液のコレクション （動画1） <ol><li>穏やかに表面殺菌に10分間、5分間、その後70％エタノールで3％、局所過酸化水素の動物や場所からダニを供給積極的に削除します。 </li><li> PAPペンでシランコーティングされた顕微鏡スライド上に円を描くとPAPペンサークル内での目盛りを配置します。 <ol><li>シランコートしたスライドは、顕微鏡スライドに体液の最高の遵守のために使用されます。 </li></ol></li><li>解剖顕微鏡（1X目的、10X接眼レンズ、3.5X倍率）の下の目盛りを表示します。 </li><li>そっとダニの足をスプレーしてダニを固定するためにピンセットでダニの背を下に押します。 </li></ol> 注：これは腸または穿刺のダニを破壊し、体液を汚染する可能性があるため、ダニを固定するときはあまりにもハードキーを押さないでください。 <ol start="5"><li>目でダニの足や足を切断使い捨てメス細かい点を持つe遠位関節。体液を介して感染を決定するために1つだけの脚は切断する必要があります。体液のコレクションのために、スライド上の複数の脚は切断されることがあります。 </li></ol> 注：これは体液の中腸の汚染を引き起こす可能性があるため、体に近すぎて足を切断しないでください。 <ol start="6"><li>脚や足が切断された後、穏やかにスライド上に足を外に分泌する体液のために目盛りの背に圧力をかけ続けています。穏やかに体液を広げるために、スライド上の周りにダニを移動します。 </li></ol> 2。唾液腺の除去 （映画2＆3） <ol><li>リン酸塩のスポットいくつかの25μlのプールは、顕微鏡スライド上に緩衝生理食塩水（PBS）とPBSプールのいずれかに目盛りを配置します。 </li><li>解剖顕微鏡（1X目的、10X接眼レンズ、3.5X倍率）の下の目盛りを表示します。 </li><li>微細な先端forcepとダニを安定させる基礎頭状花（口の部分）やダニの後部を押したままだ。 </li><li>ダニの背面に微細な先端鉗子を挿入し、臓器を公開するダニの背をスライス。必要に応じて中腸は、この時点で削除され、顕微鏡スライド上でPBSの新鮮なプールに、またはPBSを含むマイクロチューブに転送することができます。 </li><li>両側ダニの足と一緒に位置して唾液腺（ブドウのようなクラスタ）のペアを見つける。唾液腺は、ダニの破片は、唾液腺の破壊や損失を低減するためにPBSの新鮮なプールには、まだ唾液腺を含む、主要な目盛りの部分を移動する間表示されていない場合。 </li></ol> 注：彼らは、後に摂食のに比べて開発されていないため、以前の給餌では、唾液腺では見つけることが困難です。 <ol start="6"><li>微細な先端鉗子とPBSの新鮮なプール内の場所でダニから唾液腺を削除します。 </li><li>のWi細かいthは鉗子は、任意の外部の微生物やダニの残骸を削除するには、この洗浄ステップ3-4回以上繰り返し、別のきれいな25μlのPBSプールに唾液腺を転送するチップを渡した。 </li></ol> 注：個々の唾液腺の損失と混乱を減らすために静かに唾液腺クラスタを洗浄します。 <ol start="8"><li>シランコートスライド上またはPBSを含むマイクロチューブにPBSのきれいなプールに腺を配置します。 </li></ol> 3。唾液採取 （動画4） <ol><li>優しくウサギまたはそれらが完全に充血した、約5-7日後に添付ファイルをドロップオフ直前の細かい先端鉗子を使用して、他のホストから大人の女性のダニを削除します。 </li><li>スコッチテープで顕微鏡用スライドの一方の端を上にほぼ充血したダニを付着。テープは約配置する必要があります¾方法のTickの背の頭に向かって、ダニの基礎頭状花（口部分）が露出したまま。</li><li>ここで、テープが5％ピロカルピン溶液のダニ背面のピペットを5μl（メタノール中）の前縁を満たしています。テープはピロカルピンは、ダニの基礎頭状花に接触させることなく、ダニの背の上にピロカルピンを芯することができます。 </li><li>顕微鏡スライド上に非毒性粘土の作品ダニの口器から約1インチをマウントします。 </li><li>微細な先端鉗子を使用して所望の直径にプルキャピラリーチューブ28の先端を折ります。 </li><li>解剖顕微鏡下でダニの基礎頭状花を表示します。 </li><li>優しく上顎palpsは、毛細管の外側に存在することができ引っ張らキャピラリーチューブにダニの口円錐に合わせて。 </li><li>代わりに、毛細管を保持するために粘土にキャピラリーチューブの反対側の端を押してください。 </li><li>高湿度（例えば、濡れたPAPが並んで蓋付き発泡スチロールのボックスを暗室内にマウントされた垂涎の目盛りを配置する<html> ERのタオル）。重力は、唾液採取を支援することができ、口円錐指すように容器の底にスライドを傾けます。 </li><li>室温で容器を置きます。 </li><li>密接に最初の1時間は垂涎ダニを監視し、それはパスツールピペットの電球とキャピラリーチューブの外に排出することによって生成される唾液を収集します。最初の一時間後に、少なくとも4時間は蓄積時間ごとにチェックマークを付けます。それが生成される唾液を収集し続けています。 </li></ol> 注：十分な唾液が実行されて研究のために収集されると、唾液の収集が停止することができます。 <ol start="12"><li>ダニは垂涎されていない場合、または複数の唾液が必要な場合は、流涎が時折口円錐をマッサージしてキャピラリーチューブを用いて誘導することができます。 </li><li>必要になるまで-80℃で唾液や店舗℃にプロテアーゼ阻害剤カクテル0.1ボリュームを追加します。 </li></ol> 4。代表的な結果e_contentは、 &quot;&gt;ムービー1は、部分的に供給I. scapularisニンフを保持し、顕微鏡スライド上に体液を収集するために足を切断する方法を示しています。脚または足が透明な液体が（図1Aおよび1B）分泌され、切断されたら。もし腸体液はそのまま曇りが表示され切断した脚（S）（図1Cおよび1D）から出てくる破裂しています。 ニンフは、48時間または72時間のために供給された後、唾液腺の抽出は、映画の2および3に示されています。ダニ後の破片の多く（気管、マルピーギ管から成る、血液、結合組織など）がPBSの新鮮なプールに目盛りを移動する唾液腺の損失や中断を防ぐために、一般的にはありパンクである。ニンフは、オープンカットされた後に唾液腺が置かれている図2Aおよび2Bは、図2Cは、PBSのプールで唾液腺のクラスタを削除示しています。 I.から設定唾液採取scapularis成人女性I<html>のは、映画4および図3に示す。キャピラリーチューブに垂涎の目盛りは、映画5で観察される。唾液コレクションのこのメソッドは、唾液分泌を刺激すると成人女性の目盛り当たりの唾液20μlを介して得ることができますピロカルピン使用されます。 <img alt="図1。" src="/files/ftp_upload/3894/3894fig1.jpg" /> I. scapularisニンフの図1。ラベル付けされた構造。 ムービー1。 マダニscapularis体液コレクションは<a href="http://www.jove.com/files/ftp_upload/3894/3894movie1.avi">映画を見るにはここをクリックしてください</a> 。 <img alt="図2。" src="/files/ftp_upload/3894/3894fig2.jpg" /> 図2は汚染されていない（＆B）とニンフの脚から滲出（C＆D）体液を汚染。 映画（2）48時間飼育I.からの唾液腺抽出scapularisニンフ。pload/3894/3894movie2.aviは、 &quot;&gt;映画を見るにはここをクリックしてください。 72時間飼育I.からムービー3。唾液腺抽出scapularisニンフ。 <a href="http://www.jove.com/files/ftp_upload/3894/3894movie3.avi">映画を見るにはここをクリックしてください</a> 。 <img alt="図3。" src="/files/ftp_upload/3894/3894fig3.jpg" /> 図3 マダニscapularisニンフ唾液腺。 （＆B）抽出に先立って、72時間給餌ニンフの唾液腺の例は、。 （C）唾液腺のクラスタを削除しました。 大人のI.から設定ムービー4。唾液採取scapularis雌ダニ。 <a href="http://www.jove.com/files/ftp_upload/3894/3894movie4.avi">映画を見るにはここをクリックしてください</a> 。 <img alt="図4。" src="/files/ftp_upload/3894/3894fig4.jpg" /> 大人のI.から図4唾液採取scapularis女性トンicks。 （＆B）粘土によって保持されて毛細管のunpulled端とキャピラリーチューブの引き上げ最後に、その口円錐のスライド上にマウントされているチェックを入れます。よだれ大人I.を含んでいる （C＆D）加湿チャンバーscapularis女性チック。 映画5。キャピラリーチューブによだれscapularis雌ダニのマダニ 。 <a href="http://www.jove.com/files/ftp_upload/3894/3894movie5.avi">映画を見るにはここをクリックしてください</a> 。

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Entomol. 7, 193-196 (1993).</li><li>Kariu, T., Coleman, A. S., Anderson, J. F., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Rapid+Transfer+and+Localization+of+Lyme+Disease+Pathogens+Within+the+Tick+Gut.">Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut.</a> J. Vis. Exp. (48), e2544 (2011).</li><li>Edwards, K. T., Goddard, J., Varela-Stokes, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+the+internal+morphology+of+the+Ixodid+tick+Amblyomma+maculatum+koch,+(Acari:Ixodidae);+a+"How-to"+pictorial+dissection+guide.">Examination of the internal morphology of the Ixodid tick Amblyomma maculatum koch, (Acari:Ixodidae); a "How-to" pictorial dissection guide.</a> Midsouth Entomologist. 2, 28-39 (2009).</li><li>Ledin, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Borreliacidal+activity+of+saliva+of+the+tick+Amblyomma+americanum.">Borreliacidal activity of saliva of the tick Amblyomma americanum.</a> Med. Vet. Entomol. 19, 90-95 (2005).</li><li>Ribeiro, J. M., Zeidner, N. S., Ledin, K., Dolan, M. C., Mather, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+much+pilocarpine+contaminates+pilocarpine-induced+tick+saliva?.">How much pilocarpine contaminates pilocarpine-induced tick saliva?.</a> Med. Vet. Entomol. 18, 20-24 (2004).</li><li>Barker, R. W., Burris, E., Sauer, J. R., Hair, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+tick+oral+secretions+obtained+by+three+different+collection+methods.">Composition of tick oral secretions obtained by three different collection methods.</a> J. Med. Entomol. 10, 198-201 (1973).</li><li>Burgdorfer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolymph+test.+A+technique+for+detection+of+rickettsiae+in+ticks.">Hemolymph test. A technique for detection of rickettsiae in ticks.</a> Am. J. Trop. Med. Hyg. 19, 1010-1014 (1970).</li></ol>

我々は、開示することは何もありません。

ダニの体液、唾液腺、唾液のコレクションはダニ媒介性病原体の伝播の研究に重要であり、有病率、普及、増殖、目盛り、およびホスト6,11-13,20,23,29両方の持続性。目盛り30,31を分析する方法はいくつかあります。しかし、唾液腺を収集するときに唾液腺が破裂したり、ダニの遺骨が失われないように適切に目盛りを細かくすることが重要です。唾液腺は、それらが腸の汚染を...

<table border="1"><tbody><tr><td> 試薬 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>過酸化水素</td><td>フィッシャー</td><td> H312-500 </td></tr><tr><td>エタノール</td><td>アクロス</td><td> 61509-5000 </td></tr><tr><td> PBS </td><td>ボストンのバイオ製品</td><td> BM-2205 </td></tr><tr><td>デュモンファイン鉗子（3C） </td><td>フィッシャー</td><td> NC9906085 </td></tr><tr><td>シラン処理顕微鏡スライド</td><td> Bioworld </td><td> 42763007から1 </td></tr><tr><td> PAPペン</td><td> Bioworld </td><td> 21750008から1 </td></tr><tr><td>スーパーフロストプラス顕微鏡スライド</td><td>フィッシャー</td><td> 12-550-18 </td></tr><tr><td>ピロカルピン</td><td>シグマ</td><td> P6503-5G </td></tr><tr><td>プロテアーゼ阻害剤カクテル</td><td>シグマ</td><td> P2714 </td></tr><tr><td> ＃11使い捨てメス</td><td>フェザー</td><td> 2975＃11 </td></tr><tr><td>無毒粘土</td><td&gt;フィッシャー</td><td> S17307 </td></tr><tr><td>キャピラリーチューブ</td><td>チェイス科学グラス株式会社</td><td> 40A502 </td></tr></tbody></table>

saliva, salivary gland, and hemolymph collection from ixodes scapularis ticks

ダニは世界中に存在し、多くのダニ媒介性疾患を持つ人間を苦しめるされています。ダニはライム病とダニ媒介性回帰熱（ ボレリア属）、ロッキー山紅斑熱（ ロッキー山紅斑熱リケッチア ）、エーリキア症（ エールリヒアchaffeensisとE.エクイ ）、アナプラズマ病（ アナプラズマphagocytophilum）、脳炎（ダニの原因となる病原体のベクトルです。媒介性脳炎ウイルス）、バベシア症（ バベシア属）、コロラドダニ熱（Coltivirus）と、野兎病（ 野兎病菌 ）1-8。適切にホストに送信するこれらの感染性病原体は、差動、遺伝子発現を調節する目盛りのタンパク質と相互作用し、ダニ3,9-13を通して移行します。たとえば、ライム病剤、 ボレリアburgdorferiはは 、ダニの風土病サイクル14,15の祝宴と飢饉段階に差動遺伝子発現を介して適応します。また、 マダニティックとしては、消費吸血ボレリアは、唾液腺に移動すると追放された唾液9,16-19を使用してホストに送信され血体腔、腸内から複製と移行します。 ダニが供給されたホストは、通常、強力な止血及び先天性免疫応答11,13,20-22で応答します。これらのホストの応答にもかかわらず、I.ダニの唾液が3,11,20,21,23を供給するダニを支援するために免疫であるタンパク質、溶解剤、抗凝固剤、及びfibrinolysinsが含まれているため、scapularisは数日間供給できます。ダニの唾液や唾液腺抽出物（SGE）が保有する免疫調節活性は、多数のダニ媒介性病原体3,20,24-27の送信、増殖、普及を促進します。それは積極的に摂食ダニを分析し、ダニの唾液を収集することが不可欠である、さらにダニ媒介性感染症の病気を引き起こす方法を理解する。このビデオプロトコルは、解剖の技術を示しています体液の収集と積極的にIを供給から唾液腺の除去48時間および72時間後にマウスを配置した後scapularisニンフ。また、成人女性Iから唾液採取を実証scapularisダニ。

Watch this Scientific Journal Video about Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks at JoVE.com

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

ダニ媒介性病原体が病気を引き起こす方法感染したダニの体液のコレクション、唾液腺、唾液は勉強することが重要です。このプロトコルでは、餌から体液と唾液腺を収集する方法を示しています<em&gt;イクソデス·スカプラリス</em&gt;ニンフ。また、女性から唾液採取を実証<em&gt; I. scapularis</em&gt;大人。

マダニScapularisダニの唾液、唾液腺、および体液コレクション

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

saliva-salivary-gland-hemolymph-collection-from-ixodes-scapularis

Research

JoVE Journal

Immunology and Infection

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

29.2K ビュー.  Centers for Disease Control and Prevention. ダニ媒介性病原体が病気を引き起こす方法感染したダニの体液のコレクション、唾液腺、唾液は勉強することが重要です。このプロトコルでは、餌から体液と唾液腺を収集する方法を示していますイクソデス·スカプラリスニンフ。また、女性から唾液採取を実証 I. scapularis大人。

ビデオ: Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

ダニのRNA干渉

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

免疫・感染学

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

ハイブリダイゼーション<em&gt;その場で</em&gt;唾液腺、卵巣、および媒介蚊の胚の

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

ライム病研究における送信および外因診断法のために動物にダニの摂食

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

飼育<em&gt; Ixodes肩甲骨、</em&gt;ブラック・ティック・ティック：未熟期をマウスに与える

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

マウスの唾液腺機能の測定法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

マウス顎下腺唾液腺の直立した生体顕微鏡検査のための準備

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

唾液圏または単層としての体外増殖のためのヒト唾液腺からの唾液上皮細胞の単離

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

ウイルスから離れた高収率細胞外小胞製剤の精製

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

無脊椎動物の病理学研究に向けた Hemolymph のMetarhizium Blastospores の収集とRhipicephalus Microplusダニへの接種の開示

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

マウス顎下腺再生のリアルタイムライブイメージング

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

著者スポットライト:損傷後の唾液腺再生におけるマクロファージ-上皮細胞相互作用の研究 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

唾液腺における細胞間相互作用の Ex Vivo Live Cell Imaging(英語)

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...