Name: isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes
Uploaded: 2018-05-31T13:00:00
Description: Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

以下のプロトコル、マウス、研の分離を参照してください、まだ DC サブセット、他の細胞と同様に全体的な原則があります。12-16 週齢の c57bl/6 j マウスは実験動物ケアの認定-認定動物施設協会で維持されました。すべてのプロトコルは、スタンフォード大学、テルアビブ大学機関動物ケアおよび使用委員会によって承認されました。
1. 腫瘍の分離は、単球由来の DC を関?...

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マウスを予防接種に必要な DC の数が多いを与えられた (10 x 約 2-46 DC ごとに 1 つのマウス)、マウスの戦略は BM とex vivo活性化に続いて脾臓から DC の分離に基づく予防接種のほとんど。ただしアクティブ化腫瘍 DC生体内で、活性化する脾臓と BM DC の同じ条件を使用して、多くの場合、効果的な免疫の生産に成功したとされているしようとします。2 つのそれに続く出版物?...

彼らの発見以来、樹状細胞 (DC) は、T 細胞分化1をスキューするユニークな能力のための広範な研究の焦点をされています。過去数十年にわたって広範な研究努力は腫瘍の進行および免疫2中様々 な DC サブセットとその機能の定義を求めています。パターン認識受容体、組織分布の異なる異種細胞集団から成る Dc や渡り鳥と抗原提示機能3,4,5。他の DC サブセットに比べると、単球由来 DC (研) がはるかに多い腫瘍で、循環から簡単に生成することができますまたは腫瘍浸潤単球6,7。したがって、彼らの相対的な感染率の活用を求めている多くの臨床試験は、T 細胞免疫8,9を引き出す自己研の体内と体外の操作に基づいています。
同様に、実験的腫瘍モデルの DC ベースの予防接種が必要です 2-3 シリアル注射、離れて 1-2 × 10 の 5-7 日間6アクティブ DC パルスと腫瘍抗原。したがって、DC のこの大規模な数を達成するためにほとんどのマウスの研究が主に研 (IL-4 マウスの設定不要) 7-9 日間 GM-CSF の骨髄 (BM) 前駆体から培養を使用10,11。それにもかかわらず、全体的に通常 DC コンパートメント12,13マウスが GM-CSF knockout を与えられ、その文化から得られた混合集団を与えられた14これらの DC の生理学的な関連性を質問に呼ばれてきた。
また、DC は、脾臓細胞から日常的に分離することが。ただし、DC 構成は約 0.3 0.8% (約 7 × 105 DC/脾臓の結果)、合計の脾細胞と CD103 のみ、これらの細胞の+ DC と研戻るリンパ器官に移行できます。MoDCs 脾 DC 集団15,16の約 10-15% を構成する、のでほとんどの隔離のプロトコルは約 1 × 105研脾臓あたりをもたらします。研の拡大は、GM-CSF、脾研17100 増加を分泌する transfected の B16 細胞を注入することにより実現できます。しかし、研の DC ワクチンを開発するための使用は限られた人間で得られたこの手順が行われることはできませんので研は既に高度に活性化します。
DC の十分な番号を取得、に加えて自家癌細胞に対して効果的な DC ワクチンの開発の別の課題は、完全に DC をアクティブに腫瘍の設定で十分な危険信号の不足を含みます。共刺激シグナルの誘導は通常パターン認識受容体 (PRR)、または c 型レクチン シグナリング細道18,19,20,21の活性化を介して実現されます。DC の活性化のためのさらなるアプローチは、表面 Fcγ 受容体 (FcγR) との相互作用によって抗原を取る能力を悪用します。確かに、重要な写本の多くを示している予防的設定で、腫瘍の増殖を防ぐことができます BM 前駆体の活性化から研の注入腫瘍-IgG ・ IC と確立された腫瘍22,23の撲滅につながることができます.
2 つの最近の論文で、 Carmi らは、腎不全、脾 DC とは対照的血と腫瘍から研に応答できません IgG IC 追加刺激のないを発見しました。FcγR24,25をシグナル伝達を調節するチロシン脱燐酸化酵素の高い細胞内レベルの存在に起因するとこれが見つかりました。、DC の重要なチェックポイントの定義によっては、この作品は成功した DC ベースの予防接種についての重要な洞察を提供しました。FcγR シグナル伝達とおそらく同様のリン酸化カスケードを利用した他のレクチン受容体から信号を有効にする追加の刺激のための要件はこうして分離中に DC のプライミングを回避する必要性を強調します。
したがって、この議定書は研の血および骨髄および脾臓 DC から著しく異なる、腫瘍からの隔離を記述する、プロセス中に検討に値するの注意を強調します。

<table>
	<tbody>
		<tr>
			<td>Ficoll-Paque PREMIUM</td>
			<td>GE-Healthcare</td>
			<td>17-5442-02</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep</td>
			<td>StemCell Technologies</td>
			<td>07820</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 MicroBeads</td>
			<td>Miltenyi</td>
			<td>130-052-301</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19861</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Sigma</td>
			<td>C9697-50MG</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma</td>
			<td>DN25-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ThermoFisher</td>
			<td>16140071</td>
			<td>Test each lot for endotoxin</td>
		</tr>
		<tr>
			<td>PE-CD11c</td>
			<td>Biolegend</td>
			<td>117307</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-CD11b</td>
			<td>Biolegend</td>
			<td>101211</td>
			<td></td>
		</tr>
		<tr>
			<td>Brilliant Violet 650 MHCII</td>
			<td>Biolegend</td>
			<td>107641</td>
			<td></td>
		</tr>
		<tr>
			<td>AF48- CD86</td>
			<td>Biolegend</td>
			<td>105017</td>
			<td></td>
		</tr>
		<tr>
			<td>APC/Cy7-Ly-C6</td>
			<td>Biolegend</td>
			<td>108423</td>
			<td></td>
		</tr>
		<tr>
			<td>PE/Cy7-CD15</td>
			<td>Biolegend</td>
			<td>135523</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes

樹状細胞 (DC) が、細胞膜マーカー、移行パターン分布と抗原提示、T 細胞活性化能力で異なる異種細胞集団です。実験腫瘍モデルのほとんどの予防接種は、DC の数百万人を必要とするので、彼らは広く骨髄や脾臓から分離されます。ただし、これらの DC は免疫複合体 (IC) に、おそらく他の Syk 結合レクチン受容体に血および腫瘍 DC の応答から大きく異なります。重要なは、危険準の分子に DC の感度を与え、また内毒素や手順を特定の 1 つの架橋活性化受容体抗体の存在が DC のプライミングにおける結果し、パラメーター影響を及ぼします少なくとも投与量は、それらをアクティブにする必要があります。したがって、ここでは血液・腫瘍からの早期活性化を回避しながら研を分離するための詳しいプロトコルを記述します。さらに、腫瘍 IC、研の活性化とそれに続く分析のためのプロトコルが提供されます。

当初、腫瘍細胞に結合する世間知らずと同種同系マウスからの抗体能力を比較しました。このため、B16F10 LMP 腫瘍細胞はパラホルムアルデヒドで固定され広範囲洗浄します。B16F10 はもともと c57bl/6 マウス肺転移から分離された悪性黒色腫のセルラインです。LMP は KrasG12D から分離した膵腫瘍細胞/+、LSL-Trp53R172H/+、および Pdx 1 Cre マウスと 129F1 マウスで着実に成長しま?...

Watch this Scientific Journal Video about Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes at JoVE.com

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

単球由来 DC (研) は危険性関連分子のマイナーな量を感じることができるし、簡単にプライミング、したがって。血液、腫瘍、彼らの早期活性化を避けるために考慮すべき重要注意事項を強調しながら免疫複合体とその活性化から研の隔離のための詳しいプロトコルを提供します。

マウス単球由来樹状細胞とその後続の in Vitro活性化腫瘍免疫複合体との隔離のプロトコル

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Research

JoVE Journal

Cancer Research

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

結腸直腸癌モデルマウスにおける循環腫瘍細胞の単離

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

Ex Vivoひと肺腫瘍内常駐 cd8 陽性 T リンパ球の画像スライス共焦点顕微鏡を用いた

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

上に移動: シンプルかつ柔軟なIn Vitro三次元侵略試金のプロトコル

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

肉腫患者由来異種移植片からの腫瘍始発細胞の単離と特徴付け

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

濃縮と確立された皮下マウス腫瘍における腫瘍免疫と免疫のない微小のキャラクタリゼーション

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

それらの相互作用を研究する歯髄組織と癌細胞との共培養から間葉系幹細胞の分離

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

CD8 による培養腫瘍細胞のアポトーシスのリアルタイム検出+ T 細胞腫瘍浸潤骨髄細胞の免疫抑制機能を勉強するには

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

腫瘍免疫プロファイル用のマウスおよび治療応答評価における扁平上皮癌細胞の粘膜接種

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

操作された腫瘍関連好中球を腫瘍性マウスに移植し、生体内の血管新生電位を研究する

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

In Vitro Assay to Study Tumor-macrophage Interaction

腫瘍マクロファージ相互作用を研究するインビトロアッセイ

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...