Name: isolation of peritoneum-derived mast cells and their functional characterization with ca2+-imaging and degranulation assays
Uploaded: 2018-07-04T13:00:00
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。

動物のすべてのプロシージャは (カールスルーエ地域評議会によって正式に承認された) 実験動物の管理と使用のためのドイツの法律ガイドラインに従って行われました。
1. PMC 分離および腹腔内洗浄による栽培
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<td>1</td>
			<td>氷</td>
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<td>2</td>
			<td>10 mL 注射器</td>
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<td>3</td>
			<td>27 G 針</td>
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<td>4</td>
			<td>20 G ...

<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>

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 border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="943">
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	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">RPMI Medium</td>
			<td style="width:273px;">Thermo Scientific</td>
			<td style="width:212px;">21875034</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DPBS</td>
			<td>Sigma-Aldrich</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">FCS</td>
			<td>Thermo Scientific</td>
			<td>R92157</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IL-3</td>
			<td>R&#38;D Systems</td>
			<td>403-ML</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">SCF</td>
			<td>Thermo Scientific</td>
			<td>PMC2115</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Concanavalin A</td>
			<td>Sigma-Aldrich</td>
			<td>C2272</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fura-2 AM&#160;</td>
			<td>Thermo Fischer Scientific</td>
			<td>F1221</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">DNP-HSA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A6661</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Anti-DNP-Antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D-8406</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penstrep</td>
			<td>Thermo Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Compound 48/80&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C2313</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:212px;">X100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:291px;">4-Nitrophenyl 2-acetamido-2-deoxy-&#946;-D-glucopyranoside</td>
			<td>Sigma-Aldrich</td>
			<td style="width:212px;">N9376</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CoverWell Imaging Chamber</td>
			<td>Sigma-Aldrich</td>
			<td>GBL635051</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">96-Well plate, v-shaped bottom&#160;</td>
			<td>Corning</td>
			<td>3896</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">96-Well plates, flat bottom</td>
			<td>Greiner Bio-One</td>
			<td>655180</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microtiter plate reader with &#34;i-control&#34; software&#160;</td>
			<td>Tecan</td>
			<td>Nano Quant, infinite M200 Pro</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flat bottom plate&#160;</td>
			<td style="width:273px;">Greiner Bio-One</td>
			<td style="width:212px;">655180</td>
			<td></td>
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			<td height="24" style="height:24px;">Gravity fed solution application system</td>
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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 ...

These methods can help answer key questions in the field of intracellular signaling on excitable cells, such as which ion channels are critical for immune cell activation and mediator release. The main advantages of these techniques are their relative simplicity and the possibility of simultaneous analysis of large number of cells. Demonstrating Fura-2 calcium imaging in mast cells will be Kathrin Ohlenschlager, a graduate student from my laboratory.To begin this procedure, spray the euthanized mouse with 70%ethanol and fix it on a foam block with its dorsal side down using pins. Using blunt-edge scissors, remove the skin over the abdomen of the mouse and avoid damaging the peritoneal cavity. Next, inject seven milliliters of ice-cold RPMI medium in the peritoneal cavity by inserting a 27 gauge needle carefully in the peritoneum.Do not perforate any organs, and use a spot in the region of the epididymal fat to reduce the risk of an organ perforation. After injection, shake the mouse for one minute to detach the peritoneal cells into the RMPI medium. Do not shake the mouse too strongly to avoid damaging the internal organs and contaminating the peritoneal cavity with the blood.Then, reuse the syringe by equipping it with a new 20 gauge cannula. Shift the inner organs to one side by tilting the foam block and gently tapping it on the bench to make medium aspiration easier from the other side. Subsequently, insert a 20 gauge needle bevel-up to aspirate the fluid from the abdomen gently and slowly to avoid clogging by the inner organs.Collect as much fluid as possible. Then, remove the needle from the syringe and transfer the collected cell suspension in a collection tube on ice. Discard a sample tube if there is visible blood contamination.Centrifuge the tube with clean cell suspension at 300 g for five minutes. Under a sterile hood, aspirate the supernatant. Re-suspend the sample pellets in cold PMC medium and transfer the cell suspension to a 25-square-centimeter cell culture flask.Subsequently, add the growth factors IL-3 and SCF to the final concentrations of 10 nanograms per milliliter and 30 nanograms per milliliter, respectively. Incubate the cells for approximately 48 hours at 37 degrees Celsius until the next procedure. On days 12 through 15, if any experiments with stimulation of plasmalemmal IgE receptors are planned, pretreat the cells overnight with 300 nanograms per milliliter of IgE anti-DNP in standard culture medium.To prepare Fura-2 imaging, dilute Fura-2, AM stock solution in calcium HBSS to a final concentration of five micromolar in order to prepare the loading buffer. Then, mix 500 microliters of the loading buffer with 500 microliters of the PMC cell suspension. Pipette the mixture into the imaging chamber and incubate the cells for 20 to 30 minutes at room temperature in the dark.Set up the gravity-fed application system with different solutions according to the stimulation protocol. Next, mount the application system on the stage of the inverted microscope. Prepare a 40X high-aperture oil immersion objective by placing a small drop of immersion oil on it.Place the imaging chamber with the loaded cells in the application system and secure it to prevent movement during recording. Then, turn on the transmitted light and focus on the cells. Subsequently, start the imaging acquisition program in the Physiological Acquisition mode.Open the setup window and adjust the focus and the optimal acquisition time. Image a reference picture and mark the cells as ROIs. Mark the last ROI in an area where no cells are present and define it as the background.Then, complete the setup and start the measurement with a five-seconds-acquisition rate. Next, add solutions at the proper cycle number according to the design of the experiment. To measure antigen-induced elevation of intracellular calcium concentration, apply the syringe-two solution after cycle 20 and stop recording after cycle 150.To measure compound 48/80-induced elevation of intracellular calcium concentration, apply the syringe-three solution after cycle 20 and stop recording after cycle 150. To measure the elevation of intracellular calcium concentration evoked by SOCE, apply the syringe-four solution after cycle 10, apply the syringe-five solution after cycle 70, apply the syringe-four solution after cycle 120, apply the syringe-one solution after cycle 150, and stop recording after cycle 220. In the acquisition program, consecutively press the buttons Start Cutter, Mark All, Convert All, Physiology Measurements, OK, and then OK again.Save the files as tables and image stacks for offline analysis. To measure beta-hexosaminidase release, transfer 100 microliters of the cell suspension per well to a 96-well V-bottom well plate. Add 25 microliters of either stimulation or control solution to each well.Then, incubate the cells for 45 minutes at 37 degrees Celsius. Afterward, stop the reaction by placing the 96-well plate on ice for five minutes. Then, centrifuge the plate at four degrees Celsius for four minutes at 120 g.Transfer 120 microliters of the supernatant to a flat-bottom 96-well plate and place it on ice. Carefully avoid touching the cell pellets, but completely aspirate the supernatant. Next, add 125 microliters of lysis buffer to the cell pellets.Incubate the cell pellets for five minutes at room temperature and re-suspend them after the incubation by repeated pipetting. Pipette 25 microliters of the four-millimolar pNAG solution in the required wells of a new flat-bottom 96-well plate. Add 25 microliters of each supernatant and 25 microliters of each cell lysate separately to the prepared pNAG solution.Incubate the reaction batches for one hour at 37 degrees Celsius. After the incubation, pipette 150 microliters of stop solution to each well to stop the reaction. Next, analyze the plate sample light absorbance with a micro-plate reader at 405 nanometers with reference 630 nanometers for automatic background subtraction.In the acquisition program, check the box Reference, and in the absorbance program element menu, select 630 nanometers for automatic background subtraction. Beta-hexosaminidase release assay was performed to test the ability of the isolated PMCs that undergo degranulation upon crosslinking of the plasmalemmal IgE high-affinity receptors or stimulation of the Mrg PRB2 receptors. The cells were added to a V-bottom 96-well plate and stimulated according to this scheme for 45 minutes at 37 degrees Celsius.After centrifugation and removal of the supernatant, the pellets were lysed. The beta-hexosaminidase content of individual supernatants and pellet lysates was then analyzed by their reactions with pNAG during a one-hour incubation at 37 degrees Celsius. The amount of the reaction products was detected colorimetrically and the percentage of the released beta-hexosaminidase was calculated.The spontaneous release was very low, whereas the responses to strong degranulation stimuli, such as 10 micromolar ionomycin and compound 48/80 at 50 micrograms per milliliter, were over 40%and 60%respectively. Degranulation responses to DNP stimulation were dose-dependent over the concentration range of 10 to 300 milligrams per milliliter. Together, these data clearly indicate a good functional state of the isolated PMCs.Using this technique, you will obtain highly pure population of murine mature connective tissue mast cells derived from peritoneum within two weeks. While attempting this procedure, it's important to remember to maintain sterile conditions during the cell isolation and culturing to avoid bacterial contamination. Following this procedure, other methods, like electrophysiological patch-clamp ion current measurements can be performed in order to answer additional questions concerning calcium entry pathways in mast cells.After its development, Fura-2 intracellular calcium imaging technique paved the way for researchers in the field of mass cell physiology to explore calcium dependence of mast cell degranulation. After watching this video, you should have a good understanding of how to perform Fura-2 calcium imaging experiments and beta-hexosaminidase degranulation assay with mast cells. Don't forget that working with UV radiation can be hazardous for eyes and skin, and unnecessary exposure should be avoided while performing this procedure.

Research

JoVE Journal

Immunology and Infection

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

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