Name: an in vivo mouse model to measure na&#239;ve cd4 t cell activation, proliferation and th1 differentiation induced by bone marrow-derived dendritic cells
Uploaded: 2018-08-22T15:00:00
Description: Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

英語の編集を博士サイモン バートレットを感謝いたします。この調査・ デ ・ セルバンテス協会からの補助金によってサポートされたサラッド カルロス III (ISCIII) (PI14/00526;PI17/01395;CP11/00145;CPII16/00022)、ミゲル サーブレット プログラム、フンダシオン Areces;共同フォンド エウロペオ ・ デ ・開発地域 (フェダー) から資金調達。CNIC は経済省、業界競争力 (MEIC) とプロの CNIC 財団によってサポートされて、セヴェーロ オチョア Center of Excellence (SEV-2015-0505) です。RTF は、フンダシオン Areces と CNIC、ISCIII、セルバンテス de BHF で VZG によって設立された危惧地方病院 12 デ Octubre (imas12) と JMG-G ISCIII ミゲル サーブレット プログラムと imas12。

実験手順は、フンダシオン セントロ ナシオナル デによって承認された研究 Cardiovasculares カルロス III (CNIC) とスペインやヨーロッパのガイドラインに従ってコムニダード Autónoma デマドリード。マウスは特定病原体無料 (SPF) 条件で飼育し、二酸化炭素 (CO2) 吸入による安楽死されました。
1 脛骨と大腿骨からマウス骨髄細胞の分離
注: C57BL/6 コ?...

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J., Ahmed, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effector+and+memory+T-cell+differentiation:+implications+for+vaccine+development.">Effector and memory T-cell differentiation: implications for vaccine development.</a> Nature Reviews Immunology. 2 (4), 251-262 (2002).</li><li>Iborra, S., Gonzalez-Granado, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Differentiation+of+Naive+CD4(+)+T+Cells:+A+Tool+for+Understanding+the+Development+of+Atherosclerosis.">In Vitro Differentiation of Naive CD4(+) T Cells: A Tool for Understanding the Development of Atherosclerosis.</a> Methods in Molecular Biology. 1339, 177-189 (2015).</li><li>Toribio-Fernandez, 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=Lamin+A/C+augments+Th1+differentiation+and+response+against+vaccinia+virus+and+Leishmania+major.">Lamin A/C augments Th1 differentiation and response against vaccinia virus and Leishmania major.</a> Cell Death &amp; Disease. 9 (1), 9 (2018).</li><li>Lee, T., Shevchenko, I., Sprouse, M. L., Bettini, M., Bettini, M. 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このプロトコルは、BM から派生した Dc の活性化・増殖・ ナイーブ cd4 陽性 T 細胞の分化を調節する能力の評価を使用できます。また、BM から派生した Dc によって変調する CD4 T 細胞の感受性を評価するために使用もできます。このプロトコルで測定された生体内でこれらのイベントの変更ができます。
調査中の仮説によって T 細胞と DCs のいくつかの組み合わせを...

CD4 T 細胞と抗原提示細胞 (APCs) Dc などは、微生物病原体1,2に免疫の必要なメディエーターです。末梢のリンパ組織における CD4 T 細胞は Apc3,4,5によって提示された特定の抗原の認識に基づいてアクティブ化されます。活性化 cd4 陽性 T 細胞は、増殖し、正しい適応免疫応答6、7の開発に必要な個別の特定エフェクター Th 細胞に分化します。これらのプロセスの制御は、有害な組織損傷8を生成せず、病原体を殺す十分な適応性の防衛を生産するため重要です。Th 細胞は式または表面分子、転写因子やエフェクター サイトカインの生産によると定義され、病原体1への応答で不可欠な機能を実行します。Th1 細胞サブセットの細胞は、転写因子 t-bet とサイトカイン インターフェロン γ (肝腎) を表現、1細胞内の病原体に対する生体防御に参加します。ナイーブな cd4 陽性 T 細胞の活性化・増殖・ th1 細胞分化の定量化は、免疫応答における役割 T 細胞再生を評価するための有用な手段です。
このプロトコルによりin vivo解析で体外の容量には、活性化・増殖・ ナイーブ cd4 陽性 T 細胞の th1 細胞の分化を調節するための Dc の BM の派生が生成されます。プロトコルもナイーブ cd4 陽性 T 細胞活性化、増殖誘導の能力を評価して th1 細胞分化 (図 1)。この汎用性の高いプロトコルは、具体的操作またはプロトコル純粋な生体内での研究セル人口を選択できないことを回避できます。多様な分子と Dc の治療の影響は、治療または分離 BM セル9を遺伝子操作、遺伝子改変マウス5から BM を使用して学ぶことができます。同様に、養子の転送のための T 細胞の異なるソースからまたはいくつかの操作3,8,10後取得することによって T 細胞の反応を探索できます。
このプロトコルの主な利点は 2 つあります。T 細胞の活性化・増殖・ th1 細胞分化を流れの cytometry アプローチ; 分析します。これが生体内で研究、体外で発生する変更を回避したがってと細胞のタイプとだけそのまま臓器11に見られるその他の要因などを含むと組み合わされています。
重要な染料の使用は、放射能の使用を回避しながら増殖を追跡するため広く使用されている手法です。これらの試薬と拡散の測定は、細胞分裂後色素希釈法に基づいています。また、これらの染料は、複数の波長で検出することができます、複数の蛍光抗体またはマーカーと組み合わせてフローサイトメトリーによる分析が簡単に。Th1 細胞分化、増殖、活性化 T 細胞をフローサイトメトリーによる分析する方法を示すことによってこのプロトコルの有用性を強調表示します。

<table>
	<tbody>
		<tr>
			<td>Ethanol</td>
			<td>VWR Chemicals</td>
			<td>20,824,365</td>
			<td>5 L</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>14001-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11000-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>11253-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Fine Science Tools (F.S.T.)</td>
			<td>10020-00</td>
			<td>Box of 100 blades</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>SIGMA</td>
			<td>F7524</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>LONZA</td>
			<td>DE17-602E</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute medium (RPMI)</td>
			<td>GIBCO</td>
			<td>21875-034</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>25-gauge needle</td>
			<td>BD Microlance 3</td>
			<td>300600</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>Novico</td>
			<td>N15663</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tubes</td>
			<td>Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m nylon web filter</td>
			<td>BD Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood lysis buffer</td>
			<td>SIGMA</td>
			<td>R7757</td>
			<td>100 Ml</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SIGMA</td>
			<td>ED2SS-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A7906</td>
			<td>100 g</td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>SIGMA</td>
			<td>302643-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture-plates</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Cambrex</td>
			<td>BE17-737E</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Merck</td>
			<td>8-05740-0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>LONZA</td>
			<td>BE13-115E</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>LONZA</td>
			<td>BE17-605E</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine Granulocyte Macrophage colony-stimulating factor (GM-CSF)</td>
			<td>Prepotech</td>
			<td>315-03</td>
			<td></td>
		</tr>
		<tr>
			<td>lipopolysaccharide (LPS)</td>
			<td>SIGMA-ALDRICH</td>
			<td>L2018</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well-plate</td>
			<td>Costar</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>v450 anti-mouse CD11b antibody</td>
			<td>BD Biosciences</td>
			<td>560455</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD64 antibody</td>
			<td>BioLegend</td>
			<td>139303</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD115 antibody</td>
			<td>BioLegend</td>
			<td>135505</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD11c antibody</td>
			<td>BioLegend</td>
			<td>117305</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse MHCII antibody</td>
			<td>BioLegend</td>
			<td>125507</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD86 antibody</td>
			<td>BioLegend</td>
			<td>105011</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD80 antibody</td>
			<td>BioLegend</td>
			<td>104713</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry tubes</td>
			<td>Zelian</td>
			<td>300800-1</td>
			<td>PS 12X75 5mL</td>
		</tr>
		<tr>
			<td>OTII Ovoalbumine peptide</td>
			<td>InvivoGen</td>
			<td>323-339</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD8&#945; antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0081-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated IgM antibody</td>
			<td>BioLegend</td>
			<td>406503</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated B220 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0452-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD19 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0193-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated MHCII (I-Ab) antibody</td>
			<td>BioLegend</td>
			<td>115302</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11b antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0112-U500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD11c antibody</td>
			<td>BioLegend</td>
			<td>117303</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD44 antibody</td>
			<td>BioLegend</td>
			<td>103003</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>30-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse biotinylated DX5 antibody</td>
			<td>BioLegend</td>
			<td>108903</td>
			<td></td>
		</tr>
		<tr>
			<td>streptavidin-coated magnetic microbeads</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic cell separator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>QuadroMACS Separator</td>
		</tr>
		<tr>
			<td>Separation columns</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr>
			<td>PE anti-mouse CD4 antibody</td>
			<td>BioLegend</td>
			<td>100408</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD3 antibody</td>
			<td>BioLegend</td>
			<td>100235</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD8 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0081-U025</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Violet Tracer</td>
			<td>Thermofisher</td>
			<td>C34557</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD25 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0251-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa647 anti-mouse CD69 antibody</td>
			<td>BioLegend</td>
			<td>104518</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCPCY5.5 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>65-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>PECY7 anti-mouse CD45.1 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>60-0453</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD45.2 antibody</td>
			<td>Tonbo Biosciences</td>
			<td>35-0454</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>SIGMA-ALDRICH</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12 Myristate 13 Acetate (PMA)</td>
			<td>SIGMA</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>Brefeldin A (BD GolgiPlug)</td>
			<td>BD</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Millipore</td>
			<td>8-18715-02100</td>
			<td></td>
		</tr>
		<tr>
			<td>Intracellular permeabilization buffer</td>
			<td>eBioscience</td>
			<td>00-8333</td>
			<td></td>
		</tr>
		<tr>
			<td>APC anti-mouse IFNg antibody</td>
			<td>Tonbo Biosciences</td>
			<td>20-7311-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-block (anti-mouse CD16/CD32)</td>
			<td>Tonbo Biosciences</td>
			<td>70-0161-U100</td>
			<td></td>
		</tr>
		<tr>
			<td>B6.SJL CD45.1 mice</td>
			<td>The Jackson Laboratory</td>
			<td>002014</td>
			<td></td>
		</tr>
		<tr>
			<td>BD LSRFortessa&#8482; Cell Analyzer</td>
			<td>BD Biosciences</td>
			<td>649225</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI Solution</td>
			<td>Thermofisher</td>
			<td>62248</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vivo mouse model to measure na&#239;ve cd4 t cell activation, proliferation and th1 differentiation induced by bone marrow-derived dendritic cells

ナイーブな cd4 陽性 T 細胞の活性化・増殖・ T ヘルパー 1 への分化の定量化 (Th1) 細胞は免疫応答における T 細胞の役割を評価するために便利な方法です。このプロトコルでは、顆粒球マクロファージ コロニー刺激因子 (GM-CSF) の由来樹状細胞 (Dc) を取得する (BM) 骨髄前駆細胞の in vitro分化について説明します。プロトコルはまた卵白ペプチド (OVAp) の養子の転送を記述-GM 由来 CSF Dc とナイーブ cd4 陽性 T 細胞を生体内で活性化・増殖・ th1 細胞分化を解析するために OTII トランスジェニック マウスからロード、転送 CD4 T 細胞。このプロトコルは、純粋な生体内での方法の具体的操作または調査のセル人口を選択する無力によって課される制限を回避できます。さらに、このプロトコルは生体外で発生する可能性があります機能の要因など細胞の種類とそのまま臓器でのみ発見その他の要因の影響の変化を避けるため生体内の環境で研究をできます。Dc の変更を生成するための便利なツールであり、T 細胞をプロトコル適応免疫応答、潜在的起源や多数の免疫関連疾患の開発を理解するための重要な結果を提供することを変更します。

図 1は、このプロトコルで説明されている手順を示しています。図 2は、マウスの骨髄細胞の培養と分離を示しています。これらの文化に GM-CSF と LP の追加により、体外生成と dc図 3の成熟分化の流れのフローサイトメトリー解析と取得した dc OVAp ロード GM-CSF BM から派生した Dc の成熟を示して?...

Watch this Scientific Journal Video about An In Vivo Mouse Model to Measure NaÃ¯ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells at JoVE

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

今回ナイーブ cd4 陽性 T 細胞 (T 細胞) の活性化、増殖、GM-CSF 骨髄 (BM) による th1 細胞分化・生体内で定量のためのプロトコル-由来樹状細胞 (Dc)。さらに、このプロトコルでは、BM と T 細胞の分離、DC 世代と DC および T 細胞の養子移動について説明します。

メジャー ナイーブ cd4 陽性 T 細胞の活性化、増殖と骨髄由来樹状細胞による th1 細胞分化をIn Vivoマウス モデル

Quantification of na&#239;ve CD4 T cell activation, proliferation, and differentiation to T helper 1 (Th1) cells is a useful way to assess the role played ...

This method can help answer key questions in the immunology field about T-cell activation, proliferation, and Th1 differentiation, as well antigen presenting cell stimulation of adaptive immunity. The main advantage of this technique is that it allows the study of an in vivo environment while simultaneously allowing cell manipulation in vitro. Visual demonstration of this method is critical as the organ and bone isolation and adoptive transfer steps are difficult to master through text instructions alone.For bone marrow cell isolation, disinfect the hind limbs of an adult mouse with 70%ethanol and use scissors to make a lateral skin incision across the hind limbs. Insert scissors at the medial aspect of the legs, and cut the skin with the scissors until the beginning of the hind limbs. Then use forceps to separate the skin from the legs.Using scissors, separate the muscle from the femur and tibia, and use scissors to disarticulate the hip joint, breaking the femur head. Then separate the femure from the tibia without breaking the bone ends, and place the bones in a petri dish of complete medium on ice. When all of the bones have been harvested, carefully cut off the proximal and distal ends of each bone with a scalpel, and use a 1 millileter syringe equipped with a 25-guage needle to flush the bones repeatedly with a total volume of 10 millileters of warm complete RPMI medium.When all of the bones have been flushed, transfer the entire contents of the dish to a 50-millileter conical tube fitted with a 70-micrometer pore nylon web filter, and carefully dislodge any debris and cell conglomerates with gentle stirring and pipetting. Collect the bone marrow cells by centrifugation. Resuspend the pellet in one millileter of four-degree Celsius red blood cell lysis buffer with pipetting, and incubate the mix on ice for five minutes.At the end of the incubation, stop the lysis with 10 millileter of PVS, and resuspend the pellet in 25 millileters of complete medium for a second centrifugation. Then resuspend the cells in 5 millileters of fresh complete medium for counting, and centrifuge the cells again. For secondary lymphoid tissue cell isolation, collect the inguinal, axillary, brachial, cervical, and mesentaric lymph nodes, and spleen from OT-II transgenic mice, and transfer the secondary lymphoid tissues into a plastic petri dish containing 10 millileters of complete RPMI medium.When all of the tissues have been harvested, transfer the lymph nodes and spleen to a 70-micrometer cell strainer in a 50-millileter conical tube, and use a syringe plunger to homogenize the tissues. Flush the strainer with up to 15 millileters of medium, and spin down the cells by centrifugation. Then isolate the CD4-positive T cells by magnetic bead sorting, according to standard protocols.For in vivo activation of the naive OT-II CD4-positive T-cells, label the magnetic bead-isolated T-cells with vital cell tracer dye, according to standard protocols, and resuspend the labeled cells at a 10 to the sixth cells per 100 microliters of PBS concentration. Then adoptively transfer 100 microliters of cells per animal via the tail vein. Pre-warm the mice before the injection in order to ensure that the tail veins are enough dilated to allow delivery of entire cell volume.After 24 hours, deliver 10 to the fifth LPS-matured OVA peptide-loaded dendritic cells by subcutaneous injection into the foot pad of each T-cell injected animal. Two and five days after the adoptive challenge, harvest the poplitial lymph nodes from the recipient animals for tissue processing and T-cell isolation, as demonstrated. To analyze T-cell activation, stain day two-isolated cells with fluorescent antibodies against CD4, CD69, and CD25 to determine the percentage of CD64-positive, CD25-positive T-cells within the recipient CD4-positive population by flow cytometry.To assess T-cell proliferation, stain day five-isolated cells with the appropriate fluorescent antibodies against CD4, and analyze the decay of the vital cell tracer dye signal in the donor CD4-positive T-cell population by flow cytometry. To evaluate Th1 differentiation, plate four times 10 to the fifth day five-isolated T-cells per well in 200 microliters of complete medium, supplemented with ionomycin and PMA per well, in a 96-well plate. Then place the plate in a 37 degree Celsius cell culture incubator for six hours, adding Brefeldin A to each well to block cytokine secretion during the last four hours of the stimulation.At the end of the incubation, centrifuge the plate, and discard the supernatant by rapid inversion. Stain the cells with anti-CD4 antibody, followed by fixation in 100 microliters of 2%paraformaldehyde and 1%sucrose for 20 minutes at room temperature. Permeabilize the cells with 50 microliters of permeabilization buffer for 30 minutes at 4 degrees Celsius, followed by a centrifugation wash with 150 microliters of PBS per well.Next, stain for the intracellular cytokines of interest, according to standard protocols for 30 minutes at room temperature, followed by two centrifugation washes with 150 microliters of PBS supplemented with 1%saponin per well. Then, analyze the cells by flow cytometry. Flow cytometric analysis of GM-CSF bone marrow-derived dendritic cells after LPS stimulation, reveals an upregulation of maturation markers, such as MHCII, CD80, and CD86.Naive CD4 positive OT-II T-cells become activated after adoptive transfer to recipient animals by their upregulation of T-cell activation markers surface expression, and multiple rounds of cell division. Upon activation, the proliferating CD-positive OT-II T-cells demonstrate a Th1 phenotype, as demonstrated by their intracellular interferon gamma expression. Once mastered, this technique can be completed in eight hours if it's performed properly.While attempting this procedure, it's important to remember to prepare all the reagents and materials the day before the experiment. After its development, this technique paved the way for researchers in immunology to study dendritic cells and T-cells in mice. After watching this video, you should have a good understanding of how to isolate bone marrow from the femurs and tibia of mice, and how to transfer T-cells and dendritic cells to recipient animals.Don't forget that working with sharp instruments such needles, scalpels, or scissors, can be extremely hazardous, so precautions such as using the appropriate finger protection should always be taken while performing these procedures.

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

免疫・感染学

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

追跡調査のためのQdot ®二ナノとマウス骨髄由来樹状細胞の世代とラベリング

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

ナイーブCD4のアデノウイルス形質導入Tregの分化を研究するためにT細胞

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

離とナイーブCD4 Tリンパ球のTh17分化

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1感染CD4の生得センシングを評価<sup&gt; +</sup&gt;使用して形質細胞様樹状細胞によるT細胞<em&gt;エクスビボ</em&gt;共培養系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

Isolation and Intravenous Injection of Murine Bone Marrow Derived Monocytes

単離とマウス骨髄由来の単球の静脈注射

As a subtype of leukocytes and progenitors of macrophages, monocytes are involved in many important processes of organisms and are often the subject of ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

マウスナイーブCD4<sup&gt; +</sup&gt; T細胞の単離と<em&gt;インビトロ</em&gt; T細胞サブセットへの分化

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

T細胞は、樹状細胞からTransinfectionによって細菌をキャプチャ

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

Isolation and Ex Vivo Culture of V&#948;1+CD4+&#947;&#948; T Cells, an Extrathymic &#945;&#946;T-cell Progenitor

単離と<em&gt;エクスビボ</emVδ1の&gt;文化<sup&gt; +</sup&gt; CD4<sup&gt; +</sup&gt;γδT細胞、胸腺外αβT細胞前駆

The thymus, the primary organ for the generation of &#945;&#946; T cells and backbone of the adaptive immune system in vertebrates, has long been ...

In Vitro Differentiation of Human CD4+FOXP3+ Induced Regulatory T Cells (iTregs) from Na&#239;ve CD4+ T Cells Using a TGF-&#946;-containing Protocol

メジャー ナイーブ cd4 陽性 T 細胞の活性化、増殖と骨髄由来樹状細胞による th1 細胞分化をIn Vivoマウス モデル

メジャーナイーブ cd4 陽性 T 細胞の活性化、増殖と骨髄由来樹状細胞による th1 細胞分化をIn Vivoマウスモデル