Name: isolation protocol of mouse monocyte-derived dendritic cells and their subsequent in vitro activation with tumor immune complexes
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下面的协议指的是鼠标 MoDC 的隔离, 但总体原则可能也适用于其他 DC 子集单元。12-16 周大的 C57Bl/6j 小鼠被维持在美国的实验室动物 Care–accredited 动物设施认证协会。所有议定书均经斯坦福大学和特拉维夫大学机构动物护理和使用委员会批准。
1. 肿瘤相关单核细胞源性 DC 的分离
<ol><li>在层流罩中, 从 CO2安乐死小鼠中取出肿瘤, 并将肿瘤放置在没有胎牛血清的 RPMI 培?...

<ol>
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J., van der Burg, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunotherapy+of+established+(pre)malignant+disease+by+synthetic+long+peptide+vaccines.">Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines.</a> Nat Rev Cancer. 8 (5), 351-360 (2008).</li><li>Palucka, K., Banchereau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunotherapy+via+dendritic+cells.">Cancer immunotherapy via dendritic cells.</a> Nat Rev Cancer. 12 (4), 265-277 (2012).</li><li>Bol, K. F., Schreibelt, G., Gerritsen, W. R., de Vries, I. J., Figdor, C. 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H., 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=Immune+complex-loaded+dendritic+cells+are+superior+to+soluble+immune+complexes+as+antitumor+vaccine.">Immune complex-loaded dendritic cells are superior to soluble immune complexes as antitumor vaccine.</a> J Immunol. 176 (8), 4573-4580 (2006).</li><li>Carmi, Y., 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=Allogeneic+IgG+combined+with+dendritic+cell+stimuli+induce+antitumour+T-cell+immunity.">Allogeneic IgG combined with dendritic cell stimuli induce antitumour T-cell immunity.</a> Nature. 521 (7550), 99-104 (2015).</li><li>Carmi, Y., 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=Akt+and+SHP-1+are+DC-intrinsic+checkpoints+for+tumor+immunity.">Akt and SHP-1 are DC-intrinsic checkpoints for tumor immunity.</a> JCI Insight. 1 (18), e89020 (2016).</li><li>Kenkel, J. 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=An+Immunosuppressive+Dendritic+Cell+Subset+Accumulates+at+Secondary+Sites+and+Promotes+Metastasis+in+Pancreatic+Cancer.">An Immunosuppressive Dendritic Cell Subset Accumulates at Secondary Sites and Promotes Metastasis in Pancreatic Cancer.</a> Cancer Res. 77 (15), 4158-4170 (2017).</li><li>Salmon, H., 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=Expansion+and+Activation+of+CD103(+)+Dendritic+Cell+Progenitors+at+the+Tumor+Site+Enhances+Tumor+Responses+to+Therapeutic+PD-L1+and+BRAF+Inhibition.">Expansion and Activation of CD103(+) Dendritic Cell Progenitors at the Tumor Site Enhances Tumor Responses to Therapeutic PD-L1 and BRAF Inhibition.</a> Immunity. 44 (4), 924-938 (2016).</li><li>Roslansky, P. F., Novitsky, T. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+lipopolysaccharide+(LPS)+pretreatment+of+mouse+macrophages+modulates+LPS-dependent+interleukin-6+production+in+vitro.">Low-dose lipopolysaccharide (LPS) pretreatment of mouse macrophages modulates LPS-dependent interleukin-6 production in vitro.</a> Infect Immun. 64 (3), 1011-1015 (1996).</li><li>Deng, H., Maitra, U., Morris, M., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+responsible+for+the+priming+of+macrophage+activation.">Molecular mechanism responsible for the priming of macrophage activation.</a> J Biol Chem. 288 (6), 3897-3906 (2013).</li><li>Cella, 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=A+novel+inhibitory+receptor+(ILT3)+expressed+on+monocytes,+macrophages,+and+dendritic+cells+involved+in+antigen+processing.">A novel inhibitory receptor (ILT3) expressed on monocytes, macrophages, and dendritic cells involved in antigen processing.</a> J Exp Med. 185 (10), 1743-1751 (1997).</li><li>Kramer, P. 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鉴于接种疫苗的小鼠所需的 dc 数量很大 (大约 2-4 x 106 dc 每一只老鼠), 大多数小鼠的疫苗接种策略都是基于从 BM 和脾脏中分离出的 dc 和他们的前体激活。然而, 试图激活肿瘤 DC在体内, 使用相同的条件, 激活脾脏和 BM DC, 往往是不成功的产生有效的免疫力。在随后的两个出版物中, 卡米et al。发现, 血液和肿瘤 MoDC 有明显的差异, 从脾脏和 BM DC, 鉴于他们承担自然高的内在?...

自发现以来, 树突状细胞 (DC) 一直是广泛研究的焦点, 由于其独特的能力, 扭曲 T 细胞分化1。在过去几十年中, 广泛的研究工作试图定义各种 DC 子集及其在肿瘤进展和免疫中的作用2。DCs 由不同的细胞群组成, 它们在模式识别受体、组织分布和迁移和抗原表示能力方面各不相同,3,4,5。与其他 DC 子集相比, 单核细胞衍生 DC (MoDC) 在肿瘤中的丰富程度更大, 可以很容易地从循环或肿瘤浸润的单核细胞6,7中生成。因此, 许多试图利用其相对患病率的临床试验都是基于体内和体内操作自体 MoDC, 以诱导 T 细胞免疫89。
同样, 以 DC 为基础的实验性肿瘤模型接种需要2-3 系列注射, 5-7 天间隔, 1-2 x 106激活 DC 脉冲与肿瘤抗原。因此, 为了实现这一大数目的 DC, 大多数老鼠的研究主要使用 MoDC 培养的骨髓 (BM) 前体在 GM-CSF 7-9 天 (IL-4 是不需要在鼠标设置)10,11。尽管如此, 鉴于 GM-CSF 挖空小鼠有整体正常 DC 隔间12,13, 并考虑到从该区域性获得的混合种群,14这些 DC 的生理相关性已被质疑。
或者, DC 可能经常被隔离从脾脏细胞。然而, DC 只包含大约 0.3-0.8% 的总脾脏细胞 (导致大约 7 x 105 dc 或脾脏) 和这些细胞, 只有 CD103+ DC 和 MoDC 可以迁移回到淋巴器官。由于 MoDCs 包括大约10-15% 的脾脏 DC 人口15,16, 大多数隔离协议产生大约 1 x 105 MoDC 每脾脏。MoDC 的扩张可以通过注射转染 B16 细胞分泌 GM-脑脊液来实现, 导致脾脏 MoDC17增加100倍。然而, 使用 MoDC 开发 DC 疫苗是有限的, 因为这个程序不能在人类和获得的 MoDC 已经高度激活。
除了获得足够数量的 dc, 开发有效的 dc 疫苗抗自体癌细胞的另一个挑战是缺乏足够的危险信号在肿瘤的设置, 以充分激活 dc。诱导的共同刺激信号通常是通过激活模式识别受体 (PRR), 或 c 型凝集素信号通路18,19,20,21。另外一种激活 DC 的方法利用它们通过与表面 Fcγ受体 (FcγR) 的相互作用来处理抗原的能力。事实上, 一些重要的手稿表明, 注射 MoDC 的 BM 前体激活的肿瘤 IgG 芯片可以防止肿瘤生长的预防性设置, 并可导致根除已建立的肿瘤22,23.
在最近的两篇论文中,卡米等发现, 与 BMDC 和脾 DC 相反, MoDC 从血液和肿瘤不能反应的 IgG IC 没有额外的刺激。发现这是由于存在高胞内水平的酪氨酸磷酸调节 FcγR 信号24,25。通过在 dc 中定义一个关键的检查点, 这项工作提供了对成功的基于 dc 的疫苗接种要求的重要洞察力。需要额外的刺激, 以使 FcγR 信号, 并推测信号从其他凝集素受体使用类似的磷酸化级联, 从而强调了避免在其孤立的 DC 启动的必要性。
因此, 本议定书描述了从血液和肿瘤 MoDC 的分离, 这与 BM 和脾脏 DC 明显不同, 并强调了在这一过程中值得考虑的预防措施。

<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

树突状细胞 (DC) 是不同的细胞群, 它们的细胞膜标记物, 迁移模式和分布, 并在其抗原表示和 T 细胞活化能力。由于大多数实验性肿瘤模型的接种都需要数以百万计的 DC, 所以它们被广泛地从骨髓或脾脏中分离出来。然而, 这些 dc 明显不同于血液和肿瘤 DC 在他们的反应免疫复合物 (IC), 大概是其他 Syk 耦合凝集素受体。重要的是, 鉴于 dc 对危险相关分子的敏感性, 在隔离步骤之一中交联激活受体的内毒素或抗体的存在可能导致 DC 的启动, 从而影响参数, 或者至少剂量, 需要激活他们。因此, 我们在这里描述一个详细的协议, 以隔离 MoDC 从血液和肿瘤, 同时避免其过早活化。此外, 还提供了一种用于肿瘤 IC 的 MoDC 活化的协议及其后续分析。

我们最初比较了来自天真自体和同种异体小鼠的抗体与肿瘤细胞的结合能力。为此, B16F10 和 LMP 肿瘤细胞系固定在多聚甲醛和广泛洗涤。B16F10 是黑色素瘤细胞系, 最初是从 C57Bl/6 小鼠的肺转移中分离出来的。LMP 是从 KrasG12D/+、LSL-Trp53R172H/+ 和 Pdx-1-Cre 小鼠中分离出来的胰腺肿瘤细胞, 在129F1 小鼠体内生长稳定。为了获得 IC, 肿瘤细胞在冰上孵化20分钟, 每 1 x 105肿瘤细?...

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

单核细胞衍生的 DC (MoDC) 可以感觉到少量的危险相关分子, 因此很容易被引。我们提供了一个详细的协议, 以隔离 MoDC 从血液和肿瘤和他们的活化与免疫复合体, 同时强调应考虑的关键预防措施, 以避免其过早激活。

小鼠单核细胞衍生树突状细胞的分离及与肿瘤免疫配合物的后体外活化

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

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

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

科研

癌症研究

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 活体人肺肿瘤切片中居民 CD8 T 淋巴细胞的共聚焦显微成像

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

向上移动: 一个简单而灵活的体外三维入侵检测协议

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

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

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

肿瘤免疫和非免疫微环境在小鼠皮下肿瘤中的富集与鉴定

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

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

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+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

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

小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价

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

将操纵的肿瘤相关中性粒细胞转移到肿瘤携带小鼠中,以研究其血管生成潜力在Vivo

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

体外测定研究肿瘤-巨噬菌体相互作用

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