Purification of circulating plasmacytoid dendritic cells using counterflow centrifugal elutriation and immunomagnetic beads
Purification of circulating plasmacytoid dendritic cells using counterflow centrifugal elutriation and immunomagnetic beads
复制标题
DOI：
10.3109/14653249.2012.689129
复制
发表时间：
2012-08-01
期刊：
影响因子：
4.5
通讯作者：
Gregoire, Marc
中科院分区：
文献类型：
--
作者：
Coulais, Delphine;Panterne, Clarisse;Gregoire, Marc
背景目标。浆细胞样树突状细胞（Plasmacytoid dendritic cells，pDC）是一种树突状细胞（dendritic cell，DC）亚群，其特异性产生大量I型干扰素（interferon，IFN）（IFN-α，-β）以应答病毒。它们可以从外周血单核细胞（PBMC）中纯化，通常使用磁珠分选。方法.在这项研究中，我们建立了一个逆流离心淘洗（CCE）程序，以富集pDC从PBMC。我们首先使用CD 123和BDCA-2作为标记物分析每个CCE级分中pDC的存在。然后，我们使用CCE和磁珠纯化pDC，并验证它们的功能不受此过程的影响。结果通过CCE将pDC分选成含有淋巴细胞和单核细胞的那些之间的中间级分。这些中间级分中的pDC频率是PBMC中的3倍。使用负磁珠分选，从相同数量的细胞和珠子开始，我们从中间级分获得的pDC是从PBMC获得的pDC的两倍多。从PBMC和从中间级分分选的pDC的表型和IFN-α产生能力是相似的，分选后立即和用CpG-A寡脱氧核苷酸刺激后。此外，我们表明，中间级分可以冷冻保存，并在解冻后进行磁珠分选，具有相同的效率。结论.总之，我们的结果表明，CCE可以用于富集来自同一供体的淋巴细胞、单核细胞和pDC，而无需在其表面上的磁珠。我们的方法应该是有用的纯化这些细胞的实验研究，也可能适用于临床使用的免疫治疗。
Background aims. Plasmacytoid dendritic cells (pDC) are a dendritic cell (DC) subset specialized in the production of high amounts of interferon (IFN) type I (IFN-alpha, -beta) in response to viruses. They can be purified from peripheral blood mononuclear cells (PBMC), usually using magnetic bead sorting. Methods. In this study, we set up a counterflow centrifugal elutriation (CCE) procedure to enrich pDC from PBMC. We first analyzed each CCE fraction for the presence of pDC using CD123 and BDCA-2 as markers. We then purified pDC using CCE and magnetic beads and verified that their functions were not affected by this procedure. Results. pDC were sorted by CCE into intermediate fractions between those containing lymphocytes and monocytes. The pDC frequency in these intermediate fractions was 3-fold that in PBMC. Using negative-magnetic bead sorting, starting with the same number of cells and beads, we obtained more than twice as many pDC from intermediate fractions as from PBMC. The phenotypes and IFN-alpha production capacities of sorted pDC from PBMC and from intermediate fractions were similar, both immediately after sorting and after stimulation with CpG-A oligodeoxynucleotides. In addition, we showed that intermediate fractions could be cryopreserved and that magnetic bead sorting could be performed with the same efficiency after thawing. Conclusions. Altogether, our results show that CCE can be used to enrich lymphocytes, monocytes and pDC from the same donor, without magnetic beads on their surface. Our method should be useful for the purification of these cells for experimental research and may also be adaptable for clinical use in immunotherapy.