Assessing the development of murine plasmacytoid dendritic cells in Peyer's patches using adoptive transfer of hematopoietic progenitors.
Assessing the development of murine plasmacytoid dendritic cells in Peyer's patches using adoptive transfer of hematopoietic progenitors.
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DOI：
10.3791/51189
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发表时间：
2014-03-17
期刊：
影响因子：
--
通讯作者：
Watowich SS
中科院分区：
文献类型：
--
作者：
Li HS;Watowich SS
该方案详细介绍了一种分析纯化的造血祖细胞在肠道Peyer‘s结(PP)中产生浆细胞样树突状细胞(PDC)的能力的方法。用流式细胞仪从C57BL6小鼠骨髓中纯化出常见的树突状细胞前体细胞(CDPs，LIN−c-kitlo CD115+flt3+)，并将其转移给PP中缺乏大量树突状细胞前体细胞的受体小鼠，以IFNAR−/−小鼠作为转移受体。在一些小鼠中，在过继转移CDP之前，使用Flt3L编码质粒的流体动力基因转移(HGT)，促进了树突状细胞生长因子Flt3L的过度表达。Flt3L过表达可扩大来源于转移(或内源性)造血祖细胞的DC群体。在祖细胞移植后7-10天，根据CD45标记的表达，过继转移的祖细胞来源的DC与受体细胞区分开来，来自转移的CDP的PDCs为CD45.1+，而受体为CD45.2+。用流式细胞术检测受鼠PP单细胞悬液中CDP对PP中PDC数量的贡献和对Flt3L的应答能力。这种方法可以用来测试其他祖代群体是否能够产生PP-pDC。此外，这种方法可以用来研究预测影响PDC发展的因素在PP中的作用，方法是通过适当的敲除、敲除或过度表达假定的发育因子来转移祖细胞亚群和/或通过HGT操纵循环中的细胞因子。这种方法还可以分析PP pDC如何影响PPS中其他免疫亚群的频率或功能。这种方法的一个独特特点是使用了IFNAR−/−小鼠，与野生型动物相比，这些小鼠表现出严重的PP pDC的耗竭，从而允许在没有致命辐射的混杂影响的情况下重建PP pDC。
This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer's patch (PP). Common dendritic cell progenitors (CDPs, lin− c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar−/− mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7–10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar−/− mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.
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DOI：
10.1084/jem.20030107
发表时间：
2003-07-21
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
D'Amico A;Wu L
通讯作者：
Wu L
影响因子：
32.4
作者：
Goubier, Anne;Dubois, Bertrand;Gheit, Hanane;Joubert, Grgoire;Villard-Truc, Florence;Asselin-Paturel, Carine;Trinchieri, Giorgio;Kaiserlian, Dominique
通讯作者：
Kaiserlian, Dominique
影响因子：
30.5
作者：
Naik, Shalin H.;Sathe, Priyanka;Shortman, Ken
通讯作者：
Shortman, Ken
DOI：
10.1126/science.1170540
发表时间：
2009-04-17
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Liu K;Victora GD;Schwickert TA;Guermonprez P;Meredith MM;Yao K;Chu FF;Randolph GJ;Rudensky AY;Nussenzweig M
通讯作者：
Nussenzweig M
影响因子：
56.9
作者：
Fogg, DK;Sibon, C;Geissmann, F
通讯作者：
Geissmann, F