Name: selective harvesting of marginating-pulmonary leukocytes
Uploaded: 2016-03-11T15:00:00.000+00:00
Duration: 7 min 6 s
Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

著者とこの作品は、NIH / NCI助成金＃（SBE）はR01CA172138によってサポートされていました。

動物を対象とする手順は、テルアビブ大学の施設内動物管理使用委員会（IACUC）によって承認されています。 1.ラットプロトコル<ol><li>準備<ol><li> 2バタフライ21 G針を配置します。必要に応じて、針の鋭いエッジを提出することにより、2鈍いエッジの蝶21 G針を準備します。 </li><li>重力（ドライ）設定で少なくとも30分間、121℃でオートクレーブにより滅菌はさみの2ペア、平滑化エッジの鉗子、止血鉗子、歯組織鉗子：手術器具を滅菌します。 </li><li>リン酸緩衝生理食塩水（1×PBS）溶液1mlあたりヘパリン30単位を添加することにより、ヘパリン化PBS（30単位/ ml）を調製します。 RTで使用してください。 35ミリリットルは、動物ごとに必要な最小量です。 </li><li>気泡を回避するために、蠕動ポンプラインバタフライ針にヘパリン化PBSを流します。 </li></ol></li><li> MP-白血球の肺およびコレクションの灌流<ol><li> 8％のイソフルランの過剰投与により動物を安楽死させます。呼吸停止を監視することによって安楽死を確認してください。 </li><li>呼吸が停止すると、すぐに滅菌はさみや歯組織鉗子を使用して、腹膜や胸の空洞を開きます。剣状突起に腹部に沿って正中腹部切開を行います。 </li><li>あらゆる臓器や大血管を穿刺することなく、慎重に両側の肋骨を切断、ピンセットを用いて胸骨を持ち上げます。胸骨上の止血剤をクランプし、吻側心肺複合体を露出させるために胸郭を折ります。 </li><li>心臓の右心室に翼状針を挿入し、約分以内（〜250グラムの動物から6ミリリットル）をシリンジに血液の最大量を収集します。 </li><li> （ 説明を参照）は、肝臓の後方灌流を回避するために、止血剤と大静脈をクランプします。 </li><li> Pの流出管に血液を含む注射器を交換しながら、右心室の中に蝶の針を保持しますeristalticポンプ。 </li><li>心室中隔の浸透を回避し、左心室に5ミリリットルの収穫シリンジに接続された第二の翼状針を挿入します。 </li><li>約5ミリリットル/分の速度で蠕動ポンプの電源を入れ、ゆっくりとシリンジに血液で汚染された灌流液の最初のミリリットルを集めます。灌流液の色が淡赤色暗からなるまで続けます。 （血液汚染perfusteを捨てます）。 </li><li>蠕動ポンプの停止することなく、速やかに20ミリリットル収穫シリンジで5ミリリットルの収穫シリンジを交換し、灌流の高速（最大7 ml /分）を用いた肺灌流液の20ミリリットルを集めます。真空形成を回避しながら継続的に、収集シリンジへの灌流液の流れを監視します。 </li><li>蠕動ポンプの流れを止めます。 </li></ol></li><li>白血球の抽出<ol><li> 400×gで10分間の灌流液を遠心。 </li><li>流体を吸引します。 </li><li>、10 mlのPBSまたは培地を追加し、10分間400×gで遠心分離し、液体を吸引除去します。二回、この手順を繰り返します。 PBSまたは培地の使用は、サンプルの結果としての用途に依存します。 </li><li> 10分間400×gで20ミリリットルPBSまたは培地、遠心分離機を追加し、流体を吸引。 </li><li>使用例（FACS、PCR、 等 ）に基づいて、所望の濃度に細胞を再構成します。 </li></ol></li></ol> 2.マウスプロトコル<ol><li>準備<ol><li> 2バタフライ25 G針を配置します。必要に応じて、針の鋭いエッジを提出することにより、2鈍いエッジの蝶25 Gの針を準備します。 </li><li>重力（ドライ）設定で少なくとも30分間、121℃でオートクレーブにより滅菌はさみの2ペア、平滑化エッジの鉗子、止血鉗子、歯組織鉗子：手術器具を滅菌します。 </li><li> 1×PBS溶液1ml当たりヘパリンの30ユニットを追加することにより、ヘパリン化PBS（30単位/ ml）を準備します。 RTで使用してください。 25ミリリットルは、動物ごとに必要な最小量です。 </li><li> STRE気泡を回避するために、蠕動ポンプラインバタフライ針にPBSをヘパリン化しています。 </li></ol></li><li> MP-白血球の肺およびコレクションの灌流<ol><li> 8％のイソフルランの過剰投与により動物を安楽死させます。呼吸停止を監視することによって安楽死を確認してください。 </li><li>呼吸が停止すると、すぐに滅菌はさみや歯組織鉗子を使用して、腹膜や胸の空洞を開きます。剣状突起に腹部に沿って正中腹部切開を行います。 </li><li>あらゆる臓器や大血管を穿刺することなく、慎重に両側の肋骨を切断、ピンセットを用いて胸骨を持ち上げます。胸骨上の止血剤をクランプし、吻側心肺複合体を露出させるために胸郭を折ります。 </li><li> （説明を参照）は、肝臓の後方灌流を回避するために、止血剤と大静脈をクランプします。 </li><li>右ヴェンに蠕動ポンプの流出管に接続された翼状針を挿入心のtricle、および心臓の左心室、心室中隔のavoidingpenetrationに2ミリリットルの収穫シリンジに接続された第二の翼状針。 </li><li>約2ミリリットル/分の速度にポンプの電源をオンにし、灌流液は淡赤色暗からなるまで（血液汚染perfusteを捨てる）収穫シリンジに血液で汚染された灌流液の最初のミリリットルを集めます。 </li><li>蠕動ポンプの停止することなく、速やかに10ミリリットル収穫シリンジを用いて2ミリリットルの収穫シリンジを交換し、灌流の高速（最大4 ml /分）を用いた肺灌流の10ミリリットルを集めます。真空形成を回避しながら継続的に、収集シリンジへの灌流液の流れを監視します。 </li><li>蠕動ポンプの流れを止めます。 </li></ol></li><li>白血球の抽出<ol><li> 400×gで10分間の灌流液を遠心。 </li><li>流体を吸引します。 </li><li> 400で10 mlのPBSまたは培地、遠心分離機を追加10分間XG、および流体を吸引。この手順を3回繰り返します。 PBSまたは培地の使用は、サンプルの結果としての用途に依存します。 </li><li> 、10 mlのPBSまたは培地を追加し、10分間400×gで遠心分離し、液体を吸引除去します。 </li><li>使用例（FACS、PCR、 等 ）に基づいて、所望の濃度に細胞を再構成します。 </li></ol></li></ol>

<ol>
	<li>Kuebler, W. M., Goetz, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginated+pool.">The marginated pool.</a> Eur Surg Res. 34, 92-100 (2002).</li><li>Benish, M., 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=The+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunol Res. 58, 28-39 (2014).</li><li>Melamed, R., 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=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Shakhar, G., 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=Amelioration+of+operation-induced+suppression+of+marginating+pulmonary+NK+activity+using+poly+IC:+a+potential+approach+to+reduce+postoperative+metastasis.">Amelioration of operation-induced suppression of marginating pulmonary NK activity using poly IC: a potential approach to reduce postoperative metastasis.</a> Ann Surg Oncol. 14, 841-852 (2007).</li><li>Smyth, M. J., Godfrey, D. I., Trapani, 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=A+fresh+look+at+tumor+immunosurveillance+and+immunotherapy.">A fresh look at tumor immunosurveillance and immunotherapy.</a> Nat Immunol. 2, 293-299 (2001).</li><li>Talmadge, J. E., Meyers, K. M., Prieur, D. J., Starkey, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+NK+cells+in+tumour+growth+and+metastasis+in+beige+mice.">Role of NK cells in tumour growth and metastasis in beige mice.</a> Nature. 284, 622-624 (1980).</li><li>Grimm, E. A., Mazumder, A., Zhang, H. Z., Rosenberg, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphokine-activated+killer+cell+phenomenon.+Lysis+of+natural+killer-resistant+fresh+solid+tumor+cells+by+interleukin+2-activated+autologous+human+peripheral+blood+lymphocytes.">Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes.</a> J Exp Med. 155, 1823-1841 (1982).</li><li>Melamed, R., 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=Marginating+pulmonary-NK+activity+and+resistance+to+experimental+tumor+metastasis:+suppression+by+surgery+and+the+prophylactic+use+of+a+beta-adrenergic+antagonist+and+a+prostaglandin+synthesis+inhibitor.">Marginating pulmonary-NK activity and resistance to experimental tumor metastasis: suppression by surgery and the prophylactic use of a beta-adrenergic antagonist and a prostaglandin synthesis inhibitor.</a> Brain Behav Immun. 19, 114-126 (2005).</li><li>O'Dea, K. P., 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=Lung-marginated+monocytes+modulate+pulmonary+microvascular+injury+during+early+endotoxemia.">Lung-marginated monocytes modulate pulmonary microvascular injury during early endotoxemia.</a> Am J Respir Crit Care Med. 172, 1119-1127 (2005).</li><li>Sorski, L., Melamed, R., Ben-Eliyahu, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+role+for+marginating-pulmonary+and+marginating-hepatic+NK+cells+in+cancer+anti-metastatic+surveillance+and+in+immunotherapy.">A unique role for marginating-pulmonary and marginating-hepatic NK cells in cancer anti-metastatic surveillance and in immunotherapy.</a> , (2014).</li><li>Levi, B., 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=Continuous+stress+disrupts+immunostimulatory+effects+of+IL-12).">Continuous stress disrupts immunostimulatory effects of IL-12).</a> Brain Behav Immun. 25, 727-735 (2011).</li></ol>

著者らは、開示することは何もありません。

本明細書に提示された方法は、MP-白血球のユニー​​クな人口の選択的ハーベスティングと勉強を可能にします。循環または脾臓の白血球に比べて、MP集団は、白血球の亜集団の別個の組成物は、より高い活性化レベル、種々のサイトカインのより高い放出、ならびにプロおよび抗炎症性サイトカイン2,3の高いmRNAレベルによって特徴づけられます。具体的には、MP-NK細胞は、様々な標...

肺の毛細血管（ すなわち 、marginating肺（MP）白血球）1は、他の免疫の区画（ 例えば 、循環、脾臓、骨髄）からの白血球に比べて明確な白血球の組成とユニークな活性を示すことが示された、2-に付着した白血球4。例えば、循環および脾臓NK細胞、並びに分化したメッセンジャーRNA（mRNA）レベルおよびプロおよび抗炎症性サイトカインの分泌増加と比較して、種々の腫瘍細胞に対するMP-白血球展示高いナチュラルキラー（NK）細胞の細胞毒性。 MP-白血球は循環白血球（50％対それぞれ、30％）と比較して、先天性/適応免疫の高い比を有するように、細胞の組成物はまた、循環白血球から区別されます。本明細書に提示された方法の目標は、この重要かつユニークな免疫コンパートメント（細胞集団）を研究するために、およびelucに、MP-白血球の選択的ハーベスティングを可能にすることですこれらの特定の細胞上の各種操作（ 例えば 、免疫活性化）の影響をIDATE。 このユニークな集団の意義を理解するために、免疫系の細胞性免疫（CMI）のインビボでの機能を介して腫瘍細胞、微小転移、および残留疾患、循環制御することができることに留意することが重要です。この能力は、原発腫瘍を制御するために、免疫系の先行障害が発生しても明らかであり、かつ癌患者および動物モデル5 in vivoでの証拠で十分でサポートされています。重要なことには、これらの知見は、多くの場合、インビトロと矛盾しており、ほとんどの自己腫瘍細胞（細胞傷害性アッセイによって測定される）は、ヒトおよび動物から血液試料中の白血球を循環させることにより細胞毒性に抵抗性であることを報告エクスビボ研究6,7。この不一致は、前述のように異なる白血球集団のin vivoでの存在に起因し得ます前記MPの白血球集団、および活性化NK細胞3の特にその亜集団。実際に、循環および脾臓白血球に対して耐性であることが見出された同系腫瘍細胞（MADB106）は、MP-NK細胞3,8によって溶解されることが示されました。したがって、fischer344（F344）ラットの肺に転移容疑「NK抵抗性」MADB106細胞ではなく、循環または一般アクセスの容易さを与えられた研究された脾臓NK細胞によってMP-NK細胞により制御されます。 精製された、アクティブMP-白血球は肺組織の研削または生物学的分解9に基づいており、肺からの白血球の標準的な収穫方法、を介してアクセスできません。我々のアプローチは、組織処理の手法と比較して、2つの主要な利点を有します。まず、灌流アプローチは、選択的に肺実質、間質、および気管支肺胞比較例に由来する他の細胞からそれらを分離、MP-白血球を収穫しますtments。第二に、潅流技術は、より良い研削および生物学的処理が損傷細胞は、その形態を変化させることをアプローチとは異なり、完全性およびMP-白血球の生理的環境を維持し、生産および免疫活性を調節し、特にNKを抑制する様々な因子の放出を誘発します細胞毒性10。 肺は、癌転移のためにと、さまざまな感染症の主要な標的器官です。すべての循環悪性細胞と感染した細胞は、それらが変形し、毛細血管内皮細胞と常駐白血球と対話する必要が肺毛細血管を通過します。これらの条件下では、循環細胞は、容易に常駐MP-白血球によって標的化することができます。従って、この免疫コンパートメントに白血球を活性化しているように、生物学的に有利なようで、別の、生物学的実験、及び臨床現場では、このユニークなMP-人口を検討することが重要です。そのSYを注意する価値があります様々な生物学的応答調整剤によってstemic免疫活性化（ 例えば 、ポリイノシン-ポリシチジル酸（ポリI：C）またはC型CpGオリゴデオキシヌクレオチド（CPG-C ODNは））より白血球3を循環させるよりもMP-白血球を活性化することが示されています、 8,11。

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>Heparin sodium porcine mucosa (perservative free)</td><td>Sigma Aldrich</td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-pulmonary leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

MP-コンパートメント展示循環白血球に比べて異なる白血球サブセット組成物。フローサイトメトリー分析を用いて、白血球の亜集団が同定され、両方の循環及びMP白血球の組成を特徴付けるために定量化しました。顆粒球およびリンパ球は、前方および側方散乱体に基づいて同定しました。リンパ内、NKRP-1 明るい細胞は、NK細胞、単球のようなT細胞のようなC...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-pulmonary Leukocytes at JoVE.com

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating肺白血球の選択的ハーベスティング

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

selective-harvesting-of-marginating-pulmonary-leukocytes

Research

JoVE Journal

Immunology and Infection

5.8K ビュー.  Tel-Aviv University. Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

ビデオ: Selective Harvesting of Marginating-pulmonary Leukocytes

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

マウス肺胞マクロファージを採取し、無水物ナノ粒子により誘導される細胞活性化を評価する

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

生物工学

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

マウス肺樹状細胞の分離

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

免疫・感染学

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Marginating-肝白血球の選択的ハーベスティング

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

The aim of the current study is to describe a protocol for differentiating between intravascular and intraparenchymal immune cells in studies of lung inflammation. We use an intrajugular injection of a fluorescent tagged antibody prior to lung harvest. Further, we use an inflation-based lung digestion process to improve the yield of leukocytes from the lung.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of ...

Harvest of Endothelial Cells from the Balloon Tips of Swan-Ganz Catheters after Right Heart Catheterization

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further diagnose and manage PH, patients undergo repeated right heart catheterizations (RHC) wherein a Swan-Ganz catheter is advanced into a branch of the pulmonary artery and a balloon is inflated to wedge the catheter tip. This article illustrates a protocol whereby pulmonary artery endothelial cells (PAECs) may be harvested from the balloon tips of Swan-Ganz catheters after RHC, and purified with an anti- CD146 affinity column technique to purify putative PAECs. These cells might be used to provide an in situ snapshot of the biological state of the pulmonary vasculature endothelium to complement hemodynamic measurements obtained during RHC. Harvested and purified PAECs may be used for either cell culture or for subsequent analytical assays such as flow cytometery.

This protocol describes the removal and purification of pulmonary artery endothelial cells (PAECs) that adhere to the balloon tips of Swan-Ganz catheters while they are wedged in the pulmonary artery during right heart catheterization and remain adherent to the deflated balloon following removal from the patient's body.

右心カテーテル検査後 Swan-ganz カテーテルのバルーン ヒントから血管内皮細胞の収穫

A variety of pathologies lead to pulmonary hypertension (PH), which is defined as a mean pulmonary artery pressure exceeding 25 mmHg at rest. To further ...

医学

Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

マウス肺の自然免疫細胞および適応免疫細胞の同定のためのフローサイトメトリー分析

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while ...

<meta charset="utf8"></head><body>フローサイトメトリーのためのマウス肺からの免疫細胞および非免疫細胞の単離(英語)

Isolation of Live Myeloid and Epithelial Cell Populations from the Mouse Lung

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development of disease. Studies utilizing mouse models of respiratory infection, allergy, fibrosis, and cancer have been critical to reveal mechanisms of disease progression and identify therapeutic targets. However, most studies focused on the mouse lung prioritize the isolation of either immune cells or epithelial cells, rather than both populations concurrently. Here, we describe a method for preparing a comprehensive single-cell suspension of both immune and non-immune populations suitable for flow cytometry and fluorescence-activated cell sorting. These populations include epithelial cells, endothelial cells, fibroblasts, and a variety of myeloid cell subsets. This protocol entails bronchoalveolar lavage and subsequent inflation of the lungs with dispase. Lungs are then digested in a liberase mixture. This method of processing liberates a variety of diverse cell types and results in a single-cell suspension that does not require manual dissociation against a filter, promoting cell survival and yielding high numbers of live cells for downstream analyses. In this protocol, we also define gating schemes for epithelial and myeloid cell subsets in both na&#239;ve and influenza-infected lungs. Simultaneous isolation of live immune and non-immune cells is key for interrogating intercellular crosstalk and gaining a deeper understanding of lung biology in health and disease.

<meta charset="utf8"></head><body>マウス肺からの生きた骨髄系および上皮細胞集団の単離

This protocol details the isolation of live immune and non-immune populations from the mouse lung at a steady state and following influenza infection. It also provides gating strategies for identifying epithelial and myeloid cell subsets.

マウス肺からの生きた骨髄系および上皮細胞集団の単離

The lung is continuously exposed to pathogens and other noxious environmental stimuli, rendering it vulnerable to damage, dysfunction, and the development ...

磁気細胞選択法を用いたIn vitro RHEC培養法の樹立

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

著者スポットライト:軟髄膜リンパ内皮細胞の細胞機能と潜在的な臨床的意味を明らかにする 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

軟髄膜リンパ管内皮細胞の採取と初代培養

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

Marginating肺白血球の選択的ハーベスティング

要約

さらに動画を探す

この動画の章

マウス肺胞マクロファージを採取し、無水物ナノ粒子により誘導される細胞活性化を評価する

マウス肺樹状細胞の分離

Marginating-肝白血球の選択的ハーベスティング

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

右心カテーテル検査後 Swan-ganz カテーテルのバルーンヒントから血管内皮細胞の収穫

マウス肺の自然免疫細胞および適応免疫細胞の同定のためのフローサイトメトリー分析

マウス肺からの生きた骨髄系および上皮細胞集団の単離

マウス肺からの生きた骨髄系および上皮細胞集団の単離

マウス肺からの生きた骨髄系および上皮細胞集団の単離

軟髄膜リンパ管内皮細胞の採取と初代培養

Marginating肺白血球の選択的ハーベスティング

要約

さらに動画を探す

この動画の章

マウス肺胞マクロファージを採取し、無水物ナノ粒子により誘導される細胞活性化を評価する

マウス肺樹状細胞の分離

Marginating-肝白血球の選択的ハーベスティング

Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach

右心カテーテル検査後 Swan-ganz カテーテルのバルーン ヒントから血管内皮細胞の収穫

マウス肺の自然免疫細胞および適応免疫細胞の同定のためのフローサイトメトリー分析

マウス肺からの生きた骨髄系および上皮細胞集団の単離

マウス肺からの生きた骨髄系および上皮細胞集団の単離

マウス肺からの生きた骨髄系および上皮細胞集団の単離

軟髄膜リンパ管内皮細胞の採取と初代培養

右心カテーテル検査後 Swan-ganz カテーテルのバルーンヒントから血管内皮細胞の収穫