Name: myeloid innate signaling pathway regulation by malt1 paracaspase activity
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity at JoVE.com

我们感谢 elsevier 授权 (许可证编号 4334770630127) 在这里复制来自 unterreiner等人的图 2a 。(2017年)。

实验是根据诺华人类研究伦理委员会的准则和标准进行的。
1. 用人药布制备外周血单个核细胞 (pbmc)
请注意:我们在收集了50毫升的袋子里, 从健康的志愿者那里收到了一件蓬松的外套。它们是在知情同意的情况下提供的, 并通过区域间 blutspende schweizeriches rotes kreuz 收集的。除非另有说明, 否则我们在室温下使用以下程序处理这些问题。
...

<ol>
	<li>Coornaert, 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=T+cell+antigen+receptor+stimulation+induces+MALT1+paracaspase-mediated+cleavage+of+the+NF-kappaB+inhibitor+A20.">T cell antigen receptor stimulation induces MALT1 paracaspase-mediated cleavage of the NF-kappaB inhibitor A20.</a> Nature immunology. 9 (3), 263-271 (2008).</li><li>Rebeaud, F., 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=The+proteolytic+activity+of+the+paracaspase+MALT1+is+key+in+T+cell+activation.">The proteolytic activity of the paracaspase MALT1 is key in T cell activation.</a> Nature immunology. 9 (3), 272-281 (2008).</li><li>Jaworski, M., Thome, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paracaspase+MALT1:+Biological+function+and+potential+for+therapeutic+inhibition.">The paracaspase MALT1: Biological function and potential for therapeutic inhibition.</a> Cellular and Molecular Life Sciences. 73 (3), 459-473 (2016).</li><li>Meininger, I., Krappmann, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphocyte+signaling+and+activation+by+the+CARMA1-BCL10-MALT1+signalosome.">Lymphocyte signaling and activation by the CARMA1-BCL10-MALT1 signalosome.</a> Biological Chemistry. 397 (12), 1315-1333 (2016).</li><li>Qiao, Q., 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=Structural+architecture+of+the+CARMA1/Bcl10/MALT1+signalosome:+nucleation-induced+filamentous+assembly.">Structural architecture of the CARMA1/Bcl10/MALT1 signalosome: nucleation-induced filamentous assembly.</a> Molecular cell. 51 (6), 766-779 (2013).</li><li>Gross, 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=Card9+controls+a+non-TLR+signalling+pathway+for+innate+anti-fungal+immunity.">Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity.</a> Nature. 442 (7103), 651-656 (2006).</li><li>Chiffoleau, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C-type+lectin-like+receptors+as+emerging+orchestrators+of+sterile+inflammation+represent+potential+therapeutic+targets.">C-type lectin-like receptors as emerging orchestrators of sterile inflammation represent potential therapeutic targets.</a> Frontiers in Immunology. 9 (FEB), 1-9 (2018).</li><li>Taylor, P. 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=Dectin-1+is+required+for+beta-glucan+recognition+and+control+of+fungal+infection.">Dectin-1 is required for beta-glucan recognition and control of fungal infection.</a> Nature Immunology. 8 (1), 31-38 (2007).</li><li>Lanternier, F., 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=Primary+immunodeficiencies+underlying+fungal+infections.">Primary immunodeficiencies underlying fungal infections.</a> Current opinion in pediatrics. 25 (6), 736-747 (2013).</li><li>Thome, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+roles+for+MALT1+in+T-cell+activation.">Multifunctional roles for MALT1 in T-cell activation.</a> Nature reviews. Immunology. 8 (7), 495-500 (2008).</li><li>Unterreiner, A., Stoehr, N., Huppertz, C., Calzascia, T., Farady, C. J., Bornancin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+MALT1+paracaspase+inhibition+does+not+block+TNF-&#945;+production+downstream+of+TLR-4+in+myeloid+cells.">Selective MALT1 paracaspase inhibition does not block TNF-&#945; production downstream of TLR-4 in myeloid cells.</a> Immunology Letters. , 48-51 (2017).</li><li>Strasser, 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=Syk+Kinase-Coupled+C-type+Lectin+Receptors+Engage+Protein+Kinase+C-&#x3B4;+to+Elicit+Card9+Adaptor-Mediated+Innate+Immunity.">Syk Kinase-Coupled C-type Lectin Receptors Engage Protein Kinase C-&#x3B4; to Elicit Card9 Adaptor-Mediated Innate Immunity.</a> Immunity. 36 (1), 32-42 (2012).</li><li>Jaworski, 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=Malt1+protease+inactivation+efficiently+dampens+immune+responses+but+causes+spontaneous+autoimmunity.">Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.</a> The EMBO journal. 33 (23), 2765-2781 (2014).</li><li>Yu, J. W., 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=MALT1+protease+activity+is+required+for+innate+and+adaptive+immune+responses.">MALT1 protease activity is required for innate and adaptive immune responses.</a> PLoS ONE. 10 (5), 1-20 (2015).</li><li>Bardet, 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=The+T-cell+fingerprint+of+MALT1+paracaspase+revealed+by+selective+inhibition.">The T-cell fingerprint of MALT1 paracaspase revealed by selective inhibition.</a> Immunology and Cell Biology. 96 (1), 81-99 (2018).</li><li>Thoma, G., 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=Discovery+and+profiling+of+a+selective+and+efficacious+syk+inhibitor.">Discovery and profiling of a selective and efficacious syk inhibitor.</a> Journal of Medicinal Chemistry. 58 (4), 1950-1963 (2015).</li><li>Bauer, B., Steinle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HemITAM:+A+single+tyrosine+motif+that+packs+a+punch.">HemITAM: A single tyrosine motif that packs a punch.</a> Science Signaling. 10 (508), 1-10 (2017).</li><li>Gringhuis, S. I., 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=Selective+c-Rel+activation+via+Malt1+controls+anti-fungal+TH-17+immunity+by+dectin-1+and+dectin-2.">Selective c-Rel activation via Malt1 controls anti-fungal TH-17 immunity by dectin-1 and dectin-2.</a> PLoS Pathogens. 7 (1), (2011).</li><li>Dufner, A., Schamel, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+antigen+receptor-induced+activation+of+an+IRAK4-dependent+signaling+pathway+revealed+by+a+MALT1-IRAK4+double+knockout+mouse+model.">B cell antigen receptor-induced activation of an IRAK4-dependent signaling pathway revealed by a MALT1-IRAK4 double knockout mouse model.</a> Cell Communication and Signaling. 9 (1), 6 (2011).</li><li>Phelan, J. 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=A+multiprotein+supercomplex+controlling+oncogenic+signalling+in+lymphoma.">A multiprotein supercomplex controlling oncogenic signalling in lymphoma.</a> Nature. , (2018).</li></ol>

在这项工作中, 我们使用简单的实验设置来研究人和小鼠先天细胞的信号通路, 并询问他们对 alt1 蛋白溶解功能的依赖。在先前工作11的基础上, 我们的研究表明, alt1 副酶活性控制 c 型肌肽样受体诱导的细胞因子的产生, 包括 tnf-α。相反, tlr-4 诱导的 tnf-α在这两个物种中都与 alt1 无关。这些数据总体上证实了 c 型乳素样受体下游的 m异位谱的关键和选择性贡献, 早期的研究<sup class="...

2008年1,2 例发现了菌核相关淋巴组织淋巴瘤移位蛋白 1) 的副适应酶活性。自那时以来, 一些研究报告了它对淋巴细胞抗原受体反应的重要贡献。小鼠遗传模型以及药理数据支持 t 细胞、t 细胞依赖性自身免疫和 b 细胞淋巴瘤设置3,4的关键作用。在淋巴细胞中, 在标记11-bcl10-alt1 复合物5的组装时, 会发生 alt1 副酶激活, 这是由 t-或 b 细胞受体下游的抗原受体近端信号触发的。还有充分的证据表明, 类似的 card9-bcl10-大力图1复合物对于在骨髓细胞 6,7 中传播 c 型乳素样受体 (cllr) 下游的信号非常重要, 例如, 十丁-1、十丁-2 和 mincle。十分蛋白-1 已得到特别好的研究, 因为这种途径是关键的主机防御真菌感染 8,9。然而, 在 toll 样受体 (tlr) 通路中, alt1 的含义仍有争议10。最近在人类骨髓细胞中的证据排除了 alt1 副酶活性在 tlr-411 下游tnf-生产监管中的直接作用。
在本工作中, 我们利用人类和小鼠骨髓细胞的各种实验设置和刺激条件, 利用特定的药理工具抑制剂和细胞因子产生的测量, 探索先天信号通路。

<table>
	<tbody>
		<tr>
			<td>100 &#181;m nylon cell strainer</td>
			<td>Sigma</td>
			<td>CLS431752</td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml Falcon tube</td>
			<td>BD Falcon</td>
			<td>352057</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>Falcon</td>
			<td>352090</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well flat-bottom plate, with low evaporation lid</td>
			<td>Costar</td>
			<td>3595</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well V-bottom plate</td>
			<td>Costar</td>
			<td>734-1798</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Chloride - NH4Cl</td>
			<td>Sigma</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay diluent RD1-W ELISA</td>
			<td>R&#38;D</td>
			<td>895038</td>
			<td>Assay diluent</td>
		</tr>
		<tr>
			<td>Cell culture microplate, 384 well, black</td>
			<td>Greiner</td>
			<td>781986</td>
			<td></td>
		</tr>
		<tr>
			<td>Depleted Zymosan</td>
			<td>Invivogen</td>
			<td>tlrl-dzn</td>
			<td>now: tlrl-zyd</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>EDTA-Na2</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate</td>
		</tr>
		<tr>
			<td>ELISA muTNF-&#945;</td>
			<td>R&#38;D</td>
			<td>SMTA00</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440-03</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C tubes</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS dissociator</td>
			<td>MACS Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr>
			<td>GM-CSF</td>
			<td>Novartis</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat-inactivated Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10082</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>HTRF hu IL-23</td>
			<td>CisBio</td>
			<td>62HIL23PEG</td>
			<td></td>
		</tr>
		<tr>
			<td>HTRF hu TNF-&#945;</td>
			<td>CisBio</td>
			<td>62TNFPEC</td>
			<td></td>
		</tr>
		<tr>
			<td>HTRF reconstitution buffer</td>
			<td>CisBio</td>
			<td>62RB3RDE</td>
			<td>50mM Phosphate buffer, pH 7.0, 0.8M KF, 0.2% BSA</td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>R&#38;D</td>
			<td>L4516</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-4</td>
			<td>Novartis</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abbott</td>
			<td>Forene</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharides (LPS)</td>
			<td>Sigma</td>
			<td>L4391</td>
			<td>LPS used in human samples</td>
		</tr>
		<tr>
			<td>Lipopolysaccharides</td>
			<td>Sigma</td>
			<td>L4516</td>
			<td>LPS used in murine samples</td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td>Self-made</td>
			<td>-</td>
			<td>155 mM NH4Cl, 10 mM KHCO3, 1 mM EDTA, pH 7.4</td>
		</tr>
		<tr>
			<td>Magnet</td>
			<td>Stemcell</td>
			<td>18001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate, 384 well white</td>
			<td>Greiner</td>
			<td>784075</td>
			<td></td>
		</tr>
		<tr>
			<td>Monocytes enrichment kit</td>
			<td>Stemcell</td>
			<td>19059</td>
			<td></td>
		</tr>
		<tr>
			<td>Nalgene Mr. Frosty Cryo 1 &#176;C Freezing Container</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td>cooling device (containing Propanol-2)</td>
		</tr>
		<tr>
			<td>PBS 1x pH 7.4 [-] CaCl2 [-] MgCl2</td>
			<td>Gibco</td>
			<td>10010</td>
			<td>Phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>Pen/Strep</td>
		</tr>
		<tr>
			<td>Potassium bicarbonate - KHCO3</td>
			<td>Sigma</td>
			<td>P9144</td>
			<td></td>
		</tr>
		<tr>
			<td>PrestoBlue</td>
			<td>Invitrogen</td>
			<td>A13262</td>
			<td>Resazurin solution for viability assessment</td>
		</tr>
		<tr>
			<td>Propanol-2</td>
			<td>Merck</td>
			<td>1.09634</td>
			<td></td>
		</tr>
		<tr>
			<td>Read buffer</td>
			<td>MesoScale Discovery</td>
			<td>R92TC-3</td>
			<td>Tris-based buffer containing tripropylamine</td>
		</tr>
		<tr>
			<td>Recovery cell culture freezing medium</td>
			<td>Gibco</td>
			<td>12648-010</td>
			<td>freezing medium</td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute Medium (RPMI) with Glutamax</td>
			<td>Gibco</td>
			<td>61870</td>
			<td>+ 10% FBS for iMoDCs 
			+ 10% FBS + 1 mM Sodium Pyruvate + 100 U/mL Pen/Strep + 5 &#181;M &#946;-mercaptoethanol for human PBMCs and monocytes 
			+ 10% FBS + Pen/Strep + 5 &#181;M &#946;-mercaptoethanol for murine splenocytes</td>
		</tr>
		<tr>
			<td>Separation buffer</td>
			<td>Self-made</td>
			<td>-</td>
			<td>PBS pH 7.4 + 2% FBS + 1 mM EDTA pH 8.0</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco</td>
			<td>11360</td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose-6,6-dibehenate</td>
			<td>Invivogen</td>
			<td>tlrl-tdb</td>
			<td>TDB</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P7949</td>
			<td>Polysorbate 20</td>
		</tr>
		<tr>
			<td>UltraPure 0.5 M EDTA pH 8.0</td>
			<td>Invitrogen</td>
			<td>15675</td>
			<td>Ethylenediaminetetraacetic acid</td>
		</tr>
		<tr>
			<td>Viewseal sealer</td>
			<td>Greiner BioOne</td>
			<td>676070</td>
			<td></td>
		</tr>
		<tr>
			<td>V-PLEX Proinflammatory Panel 1 Human Kit</td>
			<td>MesoScale Discovery</td>
			<td>K15049D</td>
			<td>electrochemiluminescent multiplex assay (IL-1&#946;, TNF-&#945;, IL-6, IL-8)</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>31350</td>
			<td></td>
		</tr>
	</tbody>
</table>

myeloid innate signaling pathway regulation by malt1 paracaspase activity

除了在淋巴细胞中的功能外, 还有许多研究都涉及到它的功能, 副胸酶 kalt1 在模式识别受体下游的先天细胞中也发挥着重要作用。最好的研究是 c 型乳素样受体家族的 dectin-1 和 dectin-2 成员, 它们以与 alt1 相关的方式诱导 syk 和 card9 依赖信号级联, 从而导致 nf-b 激活。相比之下, toll 样受体 (tlr), 如 tlr-4, 传播 nf-b 激活, 但通过 myd88/依赖 irak 级联发出信号。尽管如此, alt1 是否可能对 tlr-4 信号做出贡献仍不清楚。作为对 falt1 副酶活性的有效和选择性抑制剂, mlt-827 的最新证据表明, 人骨髓细胞中的 tnf-ptr-4 生产与 falt1 的下游无关, 而不是 ttint-1 下游的 tnf 生产, 而 ttint-1 是丙种细胞。依赖。在这里, 我们讨论了 alt1 在模式识别传感中的选择性参与, 进一步使用了各种人和小鼠的细胞制剂, 并刺激了十丁-1、mincle 或 tlr-4 途径。我们还通过探索 tnf 之外的细胞因子, 并将 mlt-827 与 syk 抑制剂 (cpd11) 和 ikk 抑制剂 (afn700) 进行比较, 提供了更多的见解。总体而言, 这些数据为 c 型乳素样受体的 alt1 依赖性提供了进一步的证据--与 tlr 信号相比, 信号传递。

在骨髓细胞中, alt1 将激活信号传递到几种 c 型乳素样受体的下游, 如十氯化锡-1、十丁-2 和 mincle6。这些途径依赖于 (下摆) 含 itam 图案的受体 (例如, dectin-1) 或含有 itam 的共同受体 (例如, fcrγ, 用于 Dectin-1 和 mincle), 用于招募和激活 sykinase (图 1)。这导致激活蛋白激酶 c 等形, 即 pkc, 磷酸化 card9, 从而触发 card9/bcl/alt1 复合?...

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Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

alt1 调节先天免疫, 但这种情况是如何发生的仍未确定。我们使用选择性 malt1 副囊酶抑制剂 mlt-827 来解开 malt1 对 toll 样或 c 型凝集素受体下游的先天信号的贡献, 证明 malt1 调节骨髓细胞因子的产生, 并在下游c 型乳素样受体, 选择性的。

palt1 副酶活性对髓系先天信号通路的调控

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

因此，本文中的方法的目的是利用人类和小鼠系统对骨髓细胞中信号通路进行对比分析。它有助于定义所选模式识别受体的下游涉及哪个信号通路。因此，此方法易于实现。它依赖于原细胞制剂和经过验证的参考化合物，这对信号机制的阐明至关重要。演示该程序将是拉蒂巴·图伊勒和阿德琳·乌勒纳，分别是我们小组的科学家和研究员。在层流罩中，有一把无菌剪刀、一升烧杯和一个塑料袋。将先前获得的布衣放入烧杯中，然后用剪刀小心地打开。使用 25 毫米移液器添加 100 ml 的 PBS，并辅以 2 毫摩尔的 EDTA。通过缓慢向上和向下移液混合。然后将25毫升稀释的布衣转移到6个50毫升锥形离心管中，每个管都预填充了15毫升聚糖基密度梯度。在800克下将管子离心20分钟，加速度适中，无需中断，即可根据细胞密度分离细胞。离心后，将可见三层，含有红血球和粒细胞的颗粒，由血浆制作的上层，以及含有外周血单核细胞的白环。使用 10 ml 移液器收获 PBMC 环，然后将其转移到新的 50 ml 管中。用 PBS 和 EDTA 进行顶点。执行三次连续洗涤，如文本协议中所述，离心时间和速度降低。最后洗涤后，将颗粒重新在25ml的冰冷溶解缓冲液中，通过渗透压力对红血球进行解压。在室温下孵育溶液，直到溶液变得清晰。然后加入25毫升分离缓冲液，停止反应。在 150 g 下离心 8 分钟，清洗溶液。从上流液中倒出，用分离缓冲液重新浇注颗粒。计算细胞后，在培养基中稀释它们，每井12，500个细胞。将 30 ml 的电池悬浮液分配到 384 井板的每个井中。接下来，在每井中加入15个4倍的浓缩化合物溶液，并在37摄氏度下用5%的二氧化碳预孵化板一小时。如果需要，将LPS添加到每毫升1纳克的最终浓度中，或将耗尽的Zymosan加入到每毫升100微克的最终浓度中。然后在37摄氏度下孵育板，用5%的二氧化碳孵育。第二天，服用10微升的上一代来测量分泌的TNFα水平。要开始连续稀释，请用介质稀释 MLT-827 库存溶液，一次达到 8 微摩尔的浓度。使用 0.08% DMSO 的介质执行六步 1：5 串行稀释。为准备单剂量测试，稀释MLT-827、AFN700和化合物11库存溶液，一次达到4微摩尔的浓度。首先，在10毫克的贫乏的Zymosan中加入2毫升无菌无内毒素水。漩涡这种库存溶液，以均质化它。将溶液等一，然后将等分储存在 20 摄氏度。在这项研究中，人类PBMC和小鼠脾脏细胞被刺激与耗尽的Zymosan，一个德金-1激动剂，或脂质多糖，TL4激动剂。上经剂中的NTNFα释放在20小时后测量。在人类和小鼠的测定中，MLT-827化合物选择性地阻止由 Dectin-1 通路驱动的 TNF alpha 生产，而不是由 TLR4 通路驱动的 TNF alpha 生产。在使用LPS刺激的单核细胞中，TNF alpha的生产几乎完全被AFN700所废除，但它对Cpd11不敏感，这与TLR4通路对NF-kappa B活性的依赖性及其对SIC活性的依赖性是一致的。相比之下，由 Dectin-1 和 MODC 驱动的 TNF alpha 生产显示对 MLT-827、AFN700 和 Cpd11 的灵敏度。这为在 Dectin-1 通路中参与 SIC CBM 信令级联提供了进一步的证据。IL-1β、IL-6和IL-23的生产对三种抑制剂也敏感，表明其调控机制与TNF alpha相似。然而，对IL-8生产的影响有限，表明这种细胞因子具有独特的调控机制。将Trehalose-二白酸酶浓度提高至每毫升50微克以上，可产生TNF alpha、IL-6和IL-1β，根据MLT-827的阻断效应，这些β依赖于ALT1抛物酶活性。类似的结果，当挑战MODC与增加浓度耗尽的Zymosan刺激德金-1。这种方法使得能够证明抛物线马尔特1选择性地调节C型卵磷脂样受体信号。必须使用具有良好特征的参考化合物。注意库存溶液稀释过程。这对溶解度有限的化合物至关重要。

Research

JoVE Journal

Biology

Culture of myeloid dendritic cells from bone marrow precursors

文化髓系树突状细胞从骨髓前体

Myeloid dendritic cells (DCs) are frequently used to study the interactions between innate and adaptive immune mechanisms and the early response to ...

科研

生物学

Reaggregate Thymus Cultures

Reaggregate胸腺文化

Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and ...

Preparation of 2-dGuo-Treated Thymus Organ Cultures

2 dGuo治疗胸腺器官培养的制备

In the thymus, interactions between developing T-cell precursors and stromal cells that include cortical and medullary epithelial cells are known to play ...

Dissection and Staining of Drosophila Larval Ovaries

Dissection and Staining of Drosophila Larval Ovaries

夹层和染色果蝇幼虫卵巢

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

血管钙的可视化 2 +信号旁分泌源性活性氧引发

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

一个高含量的成像流程研究Grb2的信号复合体的表达克隆

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

单分子成像的基因调控<em在体内</em使用转位激活裂解（CoTrAC）

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

机械刺激引起的钙波传播的细胞单层：牛角膜内皮细胞的例子

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

代谢标记和膜分馏的比较蛋白质组学分析<em&gt;拟南芥</em&gt;悬浮细胞培养

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...