Name: isolation of peritoneum-derived mast cells and their functional characterization with ca2+-imaging and degranulation assays
Uploaded: 2018-07-04T13:00:00.000+00:00
Duration: 11 min 31 s
Description: Watch this Scientific Journal Video about Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays at JoVE.com

著者は、肥満細胞作製、培養での技術支援のジュリア Geminn を認識したいと思います。この作品は、イ共同研究センター (TR SFB) 152 によって支えられました。

<ol>
	<li>Galli, S. J., Grimbaldeston, M., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+mast+cells:+negative,+as+well+as+positive,+regulators+of+immunity.">Immunomodulatory mast cells: negative, as well as positive, regulators of immunity.</a> Nat Rev Immunol. 8 (6), 478-486 (2008).</li><li>Feyerabend, T. 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=Cre-mediated+cell+ablation+contests+mast+cell+contribution+in+models+of+antibody-+and+T+cell-mediated+autoimmunity.">Cre-mediated cell ablation contests mast cell contribution in models of antibody- and T cell-mediated autoimmunity.</a> Immunity. 35 (5), 832-844 (2011).</li><li>Razin, E., Cordon-Cardo, C., Good, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+a+pure+population+of+mouse+mast+cells+in+vitro+with+conditioned+medium+derived+from+concanavalin+A-stimulated+splenocytes.">Growth of a pure population of mouse mast cells in vitro with conditioned medium derived from concanavalin A-stimulated splenocytes.</a> Proc Natl Acad Sci U S A. 78 (4), 2559-2561 (1981).</li><li>Galli, S. J., Zsebo, K. M., Geissler, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+kit+ligand,+stem+cell+factor.">The kit ligand, stem cell factor.</a> Adv Immunol. 55, 1-96 (1994).</li><li>Galli, S. J., 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=Mast+cells+as+"tunable"+effector+and+immunoregulatory+cells:+recent+advances.">Mast cells as "tunable" effector and immunoregulatory cells: recent advances.</a> Annu Rev Immunol. 23, 749-786 (2005).</li><li>Ma, H. T., Beaven, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+Ca(2+)+signaling+in+mast+cells:+Potential+targets+for+treatment+of+mast+cell-related+diseases.">Regulators of Ca(2+) signaling in mast cells: Potential targets for treatment of mast cell-related diseases.</a> Adv Exp Med Biol. 716, 62-90 (2011).</li><li>Vig, 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=Defective+mast+cell+effector+functions+in+mice+lacking+the+CRACM1+pore+subunit+of+store-operated+calcium+release-activated+calcium+channels.">Defective mast cell effector functions in mice lacking the CRACM1 pore subunit of store-operated calcium release-activated calcium channels.</a> Nat Immunol. 9 (1), 89-96 (2008).</li><li>Freichel, M., Almering, J., Tsvilovskyy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+TRP+proteins+in+mast+cells.">The role of TRP proteins in mast cells.</a> Front Immunol. 3, 150 (2014).</li><li>Scholten, J., 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=Mast+cell-specific+Cre/loxP-mediated+recombination+in+vivo.">Mast cell-specific Cre/loxP-mediated recombination in vivo.</a> Transgenic Res. 17 (2), 307-315 (2008).</li><li>Malbec, O., 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=Peritoneal+cell-derived+mast+cells:+An+in+vitro+model+of+mature+serosal-type+mouse+mast+cells.">Peritoneal cell-derived mast cells: An in vitro model of mature serosal-type mouse mast cells.</a> J Immunol. 178 (10), 6465-6475 (2007).</li><li>Grynkiewicz, G., Poenie, M., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> J Biol Chem. 260 (6), 3440-3450 (1985).</li><li>Schwartz, L. B., Austen, K. F., Wasserman, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunologic+release+of+beta-hexosaminidase+and+beta-glucuronidase+from+purified+rat+serosal+mast+cells.">Immunologic release of beta-hexosaminidase and beta-glucuronidase from purified rat serosal mast cells.</a> J Immunol. 123 (4), 1445-1450 (1979).</li><li>Schafer, 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=Mast+cell+anaphylatoxin+receptor+expression+can+enhance+IgE-dependent+skin+inflammation+in+mice.">Mast cell anaphylatoxin receptor expression can enhance IgE-dependent skin inflammation in mice.</a> J Allergy Clin Immunol. 131 (2), 541-549 (2013).</li><li>Maurer, 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=Mast+cells+promote+homeostasis+by+limiting+endothelin-1-induced+toxicity.">Mast cells promote homeostasis by limiting endothelin-1-induced toxicity.</a> Nature. 432 (7016), 512-516 (2004).</li><li>Maurer, M., Church, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+skin+responses+induced+by+icatibant+injection+are+mast+cell+mediated+and+attenuated+by+H(1)-antihistamines.">Inflammatory skin responses induced by icatibant injection are mast cell mediated and attenuated by H(1)-antihistamines.</a> Exp Dermatol. 21 (1), 154-155 (2012).</li><li>McNeil, B. D., 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=Identification+of+a+mast-cell-specific+receptor+crucial+for+pseudo-allergic+drug+reactions.">Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.</a> Nature. 519 (7542), 237-241 (2015).</li><li>Gaudenzio, N., 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=Different+activation+signals+induce+distinct+mast+cell+degranulation+strategies.">Different activation signals induce distinct mast cell degranulation strategies.</a> J Clin Invest. 126 (10), 3981-3998 (2016).</li><li>Solis-Lopez, A., 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=Analysis+of+TRPV+channel+activation+by+stimulation+of+FCepsilonRI+and+MRGPR+receptors+in+mouse+peritoneal+mast+cells.">Analysis of TRPV channel activation by stimulation of FCepsilonRI and MRGPR receptors in mouse peritoneal mast cells.</a> PLoS One. 12 (2), 0171366 (2017).</li><li>Enerback, L., Svensson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rat+peritoneal+mast+cells+by+centrifugation+on+density+gradients+of+Percoll.">Isolation of rat peritoneal mast cells by centrifugation on density gradients of Percoll.</a> J Immunol Methods. 39 (1-2), 135-145 (1980).</li><li>Bird, G. S., DeHaven, W. I., Smyth, J. T., Putney, J. W. <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+studying+store-operated+calcium+entry.">Methods for studying store-operated calcium entry.</a> Methods. 46 (3), 204-212 (2008).</li><li>Aronson, N. N., Kuranda, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysosomal+degradation+of+Asn-linked+glycoproteins.">Lysosomal degradation of Asn-linked glycoproteins.</a> FASEB J. 3 (14), 2615-2622 (1989).</li><li>Moon, T. C., Befus, A. D., Kulka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell+mediators:+their+differential+release+and+the+secretory+pathways+involved.">Mast cell mediators: their differential release and the secretory pathways involved.</a> Front Immunol. 5, 569 (2014).</li></ol>

著者が明らかに何もありません。

MC モデルは、三菱商事の脱顆粒、走化性、接着、同様 MC 活性化に関与する細胞内のシグナル伝達経路を解明する研究を正常に使用できます。臍帯由来不死化線 (LAD2 および HMC1) など人間の MCs 使用人間 MCs のモデルいくつかの研究者は血 (CD133 陽性) 前駆細胞と末梢血前駆細胞 (CD34 陽性)。他の齧歯動物の不死化など、MC のモデル (ラット好塩基性白血病 RBL-2 H 3 MC ライン) またはプライマリ培養...

MCs は、自然免疫と獲得免疫の間に顕著な役割を果たします。具体的には、MCs は細菌や寄生虫などの病原体の殺害に参加し、潜在的に有毒の内因性ペプチドや毒 (レビュー ガリら20081を参照) のためのコンポーネントも低下します。自然免疫と適応免疫の MCs の生理的役割激論の主題であります。したがって、異なる MC 欠損マウス モデルで実施された試験で多数のデータの不一致アレルギー2MCs の免疫学的機能の体系的な再評価が必要です。成熟した MCs は、大抵集中する組織や皮膚、肺、腸などの臓器で、通常血に小さな数字でのみ発見します。MCs は MC 前駆細胞などの造血前駆細胞に由来し、分化と成熟、微小でほぼすべての血管柄付きの組織1を完了します。T 細胞由来因子インターロイキン (IL)-3 は選択的にその造血前駆細胞3からの生存・増殖・分化多能性人口マウス MCs を促進します。幹細胞因子 (SCF) は、組織の構造細胞によって生成され、MC 開発、生存、移行、および関数4の重要な役割を果たしています。個々 の MCs の特性を合成して様々 なプロテアーゼやプロテオグリカンを格納する能力によって異なります。マウス、いわゆる結合組織型 MCs は解剖学的局在、形態、およびヘパリンおよびプロテアーゼの5の内容に従って粘膜 MCs と区別されます。
MCs の増加は無料のゾル性細胞質 Ca2 + ([Ca2 +]私) が脱顆粒とエイコサノイドの生産のためだけでなく、サイトカインの合成と抗原に対する応答における転写因子の活性化は不可欠ですし、様々 な分泌刺激6。これらの刺激の主要な下流ターゲットはジアシルグリセ ロール (DAG) とイノシトール 1,4,5 三リン酸ホスファチジルイノシトール 4, 5-ビスリン酸を加水分解するホスホリパーゼ C、です。DAG はプロテインキナーゼ C を活性化し、IP3 は、Ca2 +小胞体からのイオンを解放します。これらの店の枯渇は、原形質膜 Ca2 +チャネル作動させたカルシウム エントリ (SOCE) を格納するリードを介する Ca2 +流入をアクティブにします。このプロセスは Ca2 +との相互作用を誘発-センサー、一過性受容体電位標準 (TRPC) チャネルの活性化を介してだけでなく、Orai17と小胞体で間質相互作用分子-1 (Stim1)(レビューを参照してください Freichelらのタンパク質20148) 血しょう膜の。
勉強するには、いくつかの薬理学的これらのチャネルの生理的役割、(チャンネル遮断薬の適用) または遺伝的アプローチは通常使用されます。後者の場合、タンパク質発現の抑制がターゲット mRNA (ノックダウン) によって達成されるゲノム DNA が細孔の形成 (ノックアウト) チャネルのサブユニットをコードするエクソンのグローバルまたは組織固有の9削除と編集。これらのチャンネルに十分な特異的なブロッカーの可用性は制限されています。さらに、ノックダウンの方法は、例を使用して、その有効性の注意深い制御を必要があります。 西部、ブロット分析、および多くの場合対象となるチャネル蛋白質のための特定の抗体の使用不能によって妨げられています。したがって、ノックアウト マウス モデルの使用は、そのような分析のためのゴールド スタンダードとしてみなされます。MC の機能の調査のため最寄りの in vitroモデルは分離前のヴィヴォ完全に成熟した個体としては、Pmc の栽培 (MCs体外、例えばから差別化ではなく、骨髄細胞)10。.MC 関数体外受容と β ヘキソアミダーゼの刺激を研究にも使われている骨髄由来 MCs (BMMCs) に比べてリリース 8 倍と 100 倍に増資 Pmc、それぞれ。この記事では、純粋な結合組織の数が多いを培養 2 週間タイプ MCs、さらなる分析のための十分な後に取得することができますマウス Pmc の培養と分離のメソッドについて説明します。
開発及び遺伝的コード化カルシウム指標の適用の最近の進歩にもかかわらず、使用状況限られている標的細胞に遺伝子の配信の難しさによって具体的には、一次培養 MCs など専門性の高い細胞。さらに、[Ca2 +]私の測定のための蛍光プローブのこのグループには、レシオ メトリック染料と高ダイナミック レンジまだ欠けています。これらの理由から、最寄りレシオ メトリック [Ca2 +]私の測定法はまだ蛍光染料「Fura 2」11の使用です。
現在、MC 活性化の評価のために最も一般的に使用されるアプローチと脱顆粒が β-ヘキソアミダーゼ活性の測定。Β-ヘキソアミダーゼ、アレルギーの調停は、MCs12から一定の割合でヒスタミンが分泌される MC 顆粒コンポーネントの 1 つです。Β-ヘキソアミダーゼは、マイクロ プレートの比色定量法で容易に検出できるが colorigenic 製品の測定可能な量を生成する特定の基質との反応によって容易にかつ正確に測定できます。この資料では、この様々 な刺激への応答で Pmc の脱顆粒の解析手法の応用が報告されます。
高親和性の原形質膜 IgE 受容体 (FcɛRI) の集計は MCs の分泌顆粒周囲の細胞外領域のコンテンツのリリースにつながる多彩な細胞内シグナル伝達経路をアクティブにします。特異抗原に加えて、他の多くの刺激は免疫調節メディエーター、補体によりなどの多様な配列を解放する MCs をアクティブ化できます (e.g、C3a と C5a)13、血管収縮ペプチド エンドセリン 1 (ET1)14。、数々 のカチオン性物質と pseudoallergic 反応の刺激薬として (e.g。、icatibant)15 MRGPRX216へのバインド。MRGPR 誘起 MCs 脱顆粒反応に関与する細胞内シグナル伝達経路の FcɛRI を介した細胞内シグナル伝達; と比較して特徴は不十分これらの経路は、受容体同定16後最後の数年間17の間に集中的に調査される開始。主として、カルシウム項目の後に MRGPRX2 刺激に関与する細胞膜のイオン チャネルは、理解されるに残る。したがって、本稿はまた細胞内カルシウム シグナルに焦点を当て、MRGPR アゴニストと MCs の脱顆粒が刺激されます。

<table><tbody><tr><td>RPMI Medium</td><td>Thermo Scientific</td><td>21875034</td><td></td></tr><tr><td>DPBS</td><td>Sigma-Aldrich</td><td>14190144</td><td></td></tr><tr><td>FCS</td><td>Thermo Scientific</td><td>R92157</td><td></td></tr><tr><td>IL-3</td><td>R&amp;amp;D Systems</td><td>403-ML</td><td></td></tr><tr><td>SCF</td><td>Thermo Scientific</td><td>PMC2115</td><td></td></tr><tr><td>Concanavalin A</td><td>Sigma-Aldrich</td><td>C2272</td><td></td></tr><tr><td>Fura-2 AM&amp;nbsp;</td><td>Thermo Fischer Scientific</td><td>F1221</td><td></td></tr><tr><td>Pluronic F-127</td><td>Sigma-Aldrich</td><td>P2443</td><td></td></tr><tr><td>DNP-HSA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A6661</td><td></td></tr><tr><td>Anti-DNP-Antibody&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D-8406</td><td></td></tr><tr><td>Penstrep</td><td>Thermo Scientific</td><td>15140122</td><td></td></tr><tr><td>Compound 48/80&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C2313</td><td></td></tr><tr><td>4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>4-Nitrophenyl 2-acetamido-2-deoxy-&amp;beta;-D-glucopyranoside</td><td>Sigma-Aldrich</td><td>N9376</td><td></td></tr><tr><td>CoverWell Imaging Chamber</td><td>Sigma-Aldrich</td><td>GBL635051</td><td></td></tr><tr><td>96-Well plate, v-shaped bottom&amp;nbsp;</td><td>Corning</td><td>3896</td><td></td></tr><tr><td>96-Well plates, flat bottom</td><td>Greiner Bio-One</td><td>655180</td><td></td></tr><tr><td>Microtiter plate reader with &quot;i-control&quot; software&amp;nbsp;</td><td>Tecan</td><td>Nano Quant, infinite M200 Pro</td><td></td></tr><tr><td>Flat bottom plate&amp;nbsp;</td><td>Greiner Bio-One</td><td>655180</td><td></td></tr><tr><td>Ionomycin</td><td>Sigma-Aldrich</td><td>I9657</td><td></td></tr><tr><td>50 mL Plastic Centrifuge Tubes</td><td>Sarstedt</td><td>62.547.254&amp;nbsp;</td><td></td></tr><tr><td>Culture Flasks (25 cm)</td><td>Greiner Bio-One</td><td>69160</td><td></td></tr><tr><td>Cover slips glasses round; &amp;oslash; 25 mm; No. 1</td><td>Menzel</td><td>CB00250RA1</td><td></td></tr><tr><td>Hemocytometer</td><td>VWR</td><td>631-0696</td><td></td></tr><tr><td>Inverted Microscope</td><td>Zeiss</td><td>Observer Z1</td><td></td></tr><tr><td>Objectiv Fluar 40x/1.3 Oil</td><td>Zeiss</td><td>440260-9900</td><td></td></tr><tr><td>HC Filter Set Fura 2&amp;nbsp;</td><td>AHF</td><td>H76-521</td><td></td></tr><tr><td>CCD Camera</td><td>Zeiss</td><td>AxioCam MRm&amp;nbsp;</td><td></td></tr><tr><td>Monochromatic Light Source</td><td>Sutter Instruments</td><td>Lambda DG-4</td><td></td></tr><tr><td>Imaging Acquisition Program</td><td>Zeiss</td><td>AxioVision 4.8.2&amp;nbsp;</td><td></td></tr><tr><td>Gravity fed solution application system</td><td>Custom Made</td><td>4-channel</td><td></td></tr><tr><td>15 mL Plastic Centrifuge Tubes</td><td>Sarstedt</td><td>62,554,502</td><td></td></tr><tr><td>Bench Centrifuge</td><td>Heraeus</td><td>Megafuge 1.0R</td><td></td></tr></tbody></table>

isolation of peritoneum-derived mast cells and their functional characterization with ca2+-imaging and degranulation assays

肥満細胞 (MCs)、免疫システムの一部としていくつかの病原体に対するホストを守るために、アレルギーの免疫応答の開始に重要な役割を再生します。MCs の高親和性 IgE 受容体 (受容) だけでなく、他のいくつかの受容体の刺激による IgE がバインドされた表面のクロスリンクすることにより活性化無料の細胞内 Ca2 +濃度の上昇が開始されます ([Ca2 +]私)アレルギー炎症性メディエーターの放出を促進します。これらのシグナル伝達経路に関与する分子の同定は、MC の機能制御を理解するため重要です。この記事で腹膜 MCs (Pmc) のマウスの結合組織型腹膜灌流による MCs の分離と培養のためのプロトコルについて述べる。この方法による様々 なノックアウト マウス モデルから MCs の文化は、MC シグナル伝達経路に関与するタンパク質の同定に有用なアプローチを表しています。また、Ca2 +シグナル MCs。 蛍光ベース監視の [Ca2 +]私の定量化のための重要な技術は信頼性の高い単一セル Fura 2 するイメージングのためのプロトコルについても説明し、よくするアプローチを使用Ca2 +シグナル イベント、MC の活性化にとって重要であるストア作動性カルシウム エントリを含むを研究します。MC 脱顆粒の解析、β ヘキソアミダーゼ リリース アッセイをについて説明します。MCs β-ヘキソアミダーゼで説明されているすべての 3 つ異なる分泌のサブセットの脱顆粒マーカーはで colorigenic 基板との反応によって容易に定量化ことができますはとしての β-ヘキソアミダーゼの培養液中に放出される量を。マイクロタイター プレートの比色定量法。この再現性の高い技術、コスト効果の高い特殊な装置は必要ありません。指定されたプロトコルが高収率を示します全体的にみて、典型的な MC の表面マーカーを表現する MCs の MCs の典型的な形態および表現型特徴を表示して、Ca2 +- のところ再現性の高いレスポンスを発揮イメージングおよび脱顆粒の試金。

Pmc は腹腔内洗浄によって 3 の野生型マウス (C57BL/6 n) から隔離され RPMI 培でさらに培養 1% と 20% の FCS 滅菌下で成長因子 (IL-3 10 ng/ml) と 30 ng/mL で SCF の存在下でペン球菌加湿条件37 ° C と 5% の CO2。媒体は、細胞分離後 2 と 9 の日に変更されました。細胞の形態は、伝送を用いて解析した DIC イメージングの光 (図 1A, Bを参照)。...

Watch this Scientific Journal Video about Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays at JoVE.com

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

ここでは、分離し、マウスの腹腔肥胖細胞を養うプロトコルを提案します。2 つのプロトコルの機能特性評価についても述べる: リリースされた β-ヘキソアミダーゼの比色定量に基づく細胞内 Ca2 +濃度を無料と脱顆粒試験の蛍光イメージング。

腹膜由来マスト細胞とその機能特性と Ca2 +の単離-イメージングと脱顆粒の試金

Mast cells (MCs), as a part of the immune system, play a key role in defending the host against several pathogens and in the initiation of the allergic ...

isolation-peritoneum-derived-mast-cells-their-functional

Research

JoVE Journal

Immunology and Infection

Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

12.2K ビュー.  Ruprecht-Karls Heidelberg University. ここでは、分離し、マウスの腹腔肥胖細胞を養うプロトコルを提案します。2 つのプロトコルの機能特性評価についても述べる: リリースされた β-ヘキソアミダーゼの比色定量に基づく細胞内 Ca2 +濃度を無料と脱顆粒試験の蛍光イメージング。

ビデオ: Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays

Isolation of Functional Cardiac Immune Cells

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5 These immune cells, which include mast cells, T-cells and macrophages; store and release a variety of biologically active mediators including cytokines and proteases such as tryptase.6-8 These mediators have been shown to be key players in extracellular matrix metabolism by activating matrix metalloproteinases or causing collagen accumulation by modulating the cardiac fibroblasts' function.9-11 However, available techniques for isolating cardiac immune cells have been problematic because they use bacterial collagenase to digest the myocardial tissue. This technique causes activation of the immune cells and thus a loss of function. For example, cardiac mast cells become significantly less responsive to compounds that cause degranulation.12 Therefore, we developed a technique that allows for the isolation of functional cardiac immune cells which would lead to a better understanding of the role of these cells in cardiac disease.13, 14
This method requires a familiarity with the anatomical location of the rat's xiphoid process, axilla and falciform ligament, and pericardium of the heart. These landmarks are important to increase success of the procedure and to ensure a higher yield of cardiac immune cells. These isolated cardiac immune cells can then be used for characterization of functionality, phenotype, maturity, and co-culture experiments with other cardiac cells to gain a better understanding of their interactions.

This method for isolating functional immune cells from the heart provides an alternative to the conventional methods of collagenase digestion, which causes unwanted immune cell activation, resulting in a decreased responsiveness of these cells. Our method of isolation yields functional cardiac immune cells by avoiding problems associated with enzymatic digestion.

機能心臓の免疫細胞の単離

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

免疫・感染学

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly synthesized allergic, inflammatory and immunoregulatory mediators. MCs&#8217; mediators affect multiple tissues and organs culminating in allergic and immune responses. The synthesis, storage and release of the MC mediators are highly regulated. The pre-formed mediators are packed in cytoplasmic secretory granules&#160;(SG) that fuse with the plasma membrane and release their content by regulated exocytosis. We present a protocol, based on the co-expression of a gene of interest with a reporter gene that is targeted to the SGs and is released in a regulated fashion alongside the endogenous SG mediators. The protocol enables high resolution four dimensional confocal analyses of the MC SGs and monitoring their timeline from biogenesis to triggered exocytosis. Thus, using this protocol for screening genes of interest for their phenotypic and functional impact allows deciphering the molecular mechanisms that govern the biogenesis and exocytosis of the MC SGs and identifying the regulators involved. Thereby, further insights into the cellular mechanisms that account for MCs function in health and disease should be provided.

The goal of the present&#160;protocol was to develop a method that will allow functional genomic analyses of mast cell secretion. The protocol is based on quantitative assessment of the release of a fluorescent reporter gene cotrasfected with the gene of interest and real time&#160;analyses of the secretory granule&#39;s morphology.

マスト細胞の分泌顆粒の調査。生からのエキソサイトーシスへ

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such as skin, airways and gastrointestinal tract. Best known for their detrimental role in IgE-dependent allergic reactions, MCs have also emerged as important players in host defense against venom and invading bacteria and parasites. MC phenotype and function can be influenced by microenvironmental factors that may differ according to anatomic location and/or based on the type or stage of development of immune responses. For this reason, we and others have favored in vivo approaches over in vitro methods to gain insight into MC functions. Here, we describe methods for the generation of mouse bone marrow-derived cultured MCs (BMCMCs), their adoptive transfer into genetically MC-deficient mice, and the analysis of the numbers and distribution of adoptively transferred MCs at different anatomical sites. This method, named the &#8216;mast cell knock-in&#8217; approach, has been extensively used over the past 30 years to assess the functions of MCs and MC-derived products in vivo. We discuss the advantages and limitations of this method, in light of alternative approaches that have been developed in recent years.

Analyzing the Functions of Mast Cells In Vivo Using 'Mast Cell Knock-in' Mice

We describe a method for the generation of in vitro derived mast cells, their engraftment into mast cell-deficient mice, and the analysis of the phenotype, numbers and distribution of engrafted mast cells at different anatomical sites. This protocol can be used to assess the functions of mast cells in vivo.

「マスト細胞ノックイン」マウスを用いた生体内肥満細胞の機能解析

Mast cells (MCs) are hematopoietic cells which reside in various tissues, and are especially abundant at sites exposed to the external environment, such ...

Assessment of Perigenital Sensitivity and Prostatic Mast Cell Activation in a Mouse Model of Neonatal Maternal Separation

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the age of 50, yet it is the least understood and studied chronic pelvic pain disorder. A significant subset of patients with chronic pelvic pain report having experienced early life stress or abuse, which can markedly affect the functioning and regulation of the hypothalamic-pituitary-adrenal (HPA) axis. Mast cell activation, which has been shown to be increased in both urine and expressed prostatic secretions of CP/CPPS patients, is partially regulated by downstream activation of the HPA axis. Neonatal maternal separation (NMS) has been used for over two decades to study the outcomes of early life stress in rodent models, including changes in the HPA axis and visceral sensitivity. Here we provide a detailed protocol for using NMS as a preclinical model of CP/CPPS in male C57BL/6 mice. We describe the methodology for performing NMS, assessing perigenital mechanical allodynia, and histological evidence of mast cell activation. We also provide evidence that early psychological stress can have long-lasting effects on the male urogenital system in mice.

We are measuring perigenital mechanical sensitivity and mast cell activation in the prostate of male C57BL/6 mice that underwent an early life stress paradigm &#8211; neonatal maternal separation, in order to induce a preclinical model of chronic prostatitis/chronic pelvic pain syndrome.

新生児母子分離のマウスモデルにおけるPerigenital感度と前立腺肥満細胞の活性化の評価

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) has a lifetime prevalence of 14% and is the most common urological diagnosis for men under the ...

医学

An Ex vivo Mast Cell Degranulation Assay using Crude Peritoneal Exudate Cells and Natural Antigen Stimulation

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the effect of a pharmacological agent of interest on mast cells in vivo. As the anaphylactic symptoms are primarily attributed to exocytosis of the granules from mast cells, it is conceived that the agent to cause amelioration of the symptoms has a mast cell stabilizing activity. Despite the fact, it is prudent to confirm the activity by directly demonstrating the decline in the functional activity of mast cells following its treatment. In vitro degranulation assays using an immortalized mast cell line or cultured primary mast cells are routinely employed to that end. The results from the in vitro and in vivo assays may not always be akin to each other; however, as treatment conditions (e.g., treatment dose, time, surrounding environments) for the in vitro assays are often distinct from those for the in vivo assay such as PSA. In pursuit of an in vitro (or ex vivo) assay to reflect more closely the effect of a pharmacological agent on mast cells in vivo, we devised the ex vivo mast cell degranulation assay in which crude peritoneal exudate cells (PECs) isolated from the mice, treated with the agent and administered anti-dinitrophenol (DNP) IgE, were incubated directly with DNP on a carrier protein. It turned out that the assay was not only useful in validating the mast cell stabilizing activity of a pharmacological agent indicated by the in vivo assay but also practical and highly reproducible.

We have established an ex vivo mast cell degranulation assay carried out by incubating crude peritoneal exudate cells isolated from the mice, treated with a pharmacological agent of interest and administered anti-dinitrophenol (DNP) IgE beforehand, with DNP on a carrier protein.

Mast cell stabilizers are an essential part of allergy medication. Passive systemic anaphylaxis (PSA) is an animal assay widely used for investigating the ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

工学的HEK細胞を用いたMRGPRX2を活性化するペプチドのスクリーニング

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

そのままに、全体のマウス乳腺の腺の形態と細胞外マトリックス式の分析のための分離

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

生物学

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

成人ネズミ涙腺および顎下腺からの筋上皮細胞の単離

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...

Mast Cell Degranulation Assay to Study the Effect of a Target Chemical

Source: Tsvilovskyy, V., et al. <a href="https://app.jove.com/v/57222">Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays.</a>&#160;J. Vis. Exp. (2018).
This video demonstrates a colorimetric assay to determine the degranulation activity of mast cells. The enzyme released by the mast cells hydrolyzes the substrate and produces a colored product. The degranulation activity of the mast cells was measured as the percentage release of enzymes outside the cell normalized with the total enzyme content in the cell.

標的化学物質の効果を研究するための肥満細胞脱顆粒アッセイ

Source: Tsvilovskyy, V., et al. Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation ...

JoVE Encyclopedia of Experiments

免疫学

免疫システムとコンポーネント

アッセイと機能プロファイリング

A Technique to Isolate Primary Mast Cells from a Murine Peritoneal Cavity

Source: Tsvilovskyy, V., et al. <a href="https://app.jove.com/v/57222">Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays.</a> J. Vis. Exp. (2018).
This video demonstrates a peritoneal lavage method for isolating mature connective tissue-type mast cells from the peritoneal cavity of adult mice. Upon isolating a mixture of primary immune cells from the peritoneal cavity, selective proliferation of mast cells is performed under appropriate culture conditions.

腹膜由来マスト細胞とその機能特性と Ca^{2 +}の単離-イメージングと脱顆粒の試金

要約

さらに動画を探す

この動画の章