Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: a lesson learned from flow cytometry.
Autofluorescence contributes to false-positive intracellular Foxp3 staining in macrophages: a lesson learned from flow cytometry.
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DOI：
10.1016/j.jim.2012.08.014
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发表时间：
2012-12-14
影响因子：
2.2
通讯作者：
Sharifi, Behrooz G.
中科院分区：
文献类型：
--
作者：
Li, Fuqiang;Yang, Mingjie;Wang, Lai;Williamson, Ian;Tian, Fang;Qin, Minghui;Shah, Prediman K.;Sharifi, Behrooz G.
叉头盒P3（Foxp 3）因其在调节性T细胞（T细胞）中的高度限制性表达而众所周知。最近的一项研究表明，在小鼠骨髓、脾脏、肝脏、淋巴结和胸腺中存在Foxp 3阳性巨噬细胞亚群，其表现出类似于TclD的免疫调节作用。在这篇报道被撤回之前，我们试图研究这种巨噬细胞亚群在高脂血症小鼠模型中的功能。从C57 BL/6 apo E−/−小鼠分离的骨髓和脾细胞用抗CD 11b、抗F4/80和抗Foxp 3染色，并通过流式细胞术进行分析。结果表明，骨髓和脾脏中Foxp 3阳性率分别为3.06-8.08%和0.24-2.21%。出乎意料的是，未染色或同种型染色的对照也显示出强烈的自体荧光，并且这些细胞的相似百分比落入计数抗Foxp 3染色群体的相同FL 1通道内。SSC/FSC图上的自发荧光群体的反向门控显示该细胞群体具有较高的侧向散射。腹腔巨噬细胞（PMH）也显示出类似的自发荧光。我们使用qPCR进一步评估用M-CSF、M-CSF+IL-4、M-CSF+TGFβ1处理的PMM 1或在存在抗CD 3和CD 28抗体共刺激物的情况下用TGFβ1处理的BMDM中Foxp 3 mRNA的表达。在两种细胞培养系统中均未检测到Foxp 3 mRNA的表达，而在用TGFβ1刺激的幼稚CD 4+细胞中诱导了稳健的Foxp 3基因表达。与这些发现相一致，荧光显微镜显示在PMS 2中没有Foxp 3蛋白表达，然而在诱导的TCL 2中很容易检测到Foxp 3表达。我们的结论是，报道的Foxp 3在巨噬细胞中的表达很可能是一个伪像，严格的多模态方法是证明候选基因在任何细胞类型中表达的关键。
Forkhead box P3 (Foxp3) is well known for its highly restricted expression in T regulatory cells (Tregs). A recent study suggested the existence of a Foxp3 positive macrophage subpopulation in mouse bone marrow, spleen, liver, lymph nodes, and thymus that exhibited immune regulatory effect similar to Tregs. Before this report was retracted, we attempted to study the function of this macrophage subpopulation in a mouse model of hyperlipidemia. Bone marrow and spleen cells isolated from C57BL/6 apo E−/− mice were stained with anti-CD11b, anti-F4/80 and anti-Foxp3 and analyzed by flow cytometry. Our results showed that 3.06–8.08% of CD11b+F4/80+ macrophages from bone marrow cells and 0.24–2.21% from splenic were Foxp3-positive. Unexpectedly, unstained or isotype stained controls also showed strong autofluorescence and similar percentages of these cells fell within the same FL1 channel that counted the anti-Foxp3 stained population. Back gating of the autofluorescent population onto a SSC/FSC plot showed that this population of cells had a higher side scatter. The peritoneal macrophages (PMø) exhibited similar autofluorescence. We used qPCR to further evaluate the expression of Foxp3 mRNA in PMø that were treated with M-CSF, M-CSF+IL-4, M-CSF+TGFβ1 or in BMDM treated with TGFβ1 in the presence of anti-CD3 and CD28 antibody co-stimulators. No expression of Foxp3 mRNA was detected in either cell culture systems, whereas robust Foxp3 gene expression was induced in naïve CD4+ cells stimulated with TGFβ1. Consistent with these findings, fluorescence microscopy showed no Foxp3 protein expression in PMø, however Foxp3 expression was easily detected in induced Tregs. We conclude that the reported expression of Foxp3 in macrophages is likely an artifact and that a stringent multimodality approach is critical to demonstrate candidate gene expression in any cell type.
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影响因子：
14.8
作者：
Fantini, Massimo C.;Dominitzki, Sabine;Becker, Christoph
通讯作者：
Becker, Christoph
DOI：
10.1084/jem.20100730
发表时间：
2011-07-04
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Manrique SZ;Correa MA;Hoelzinger DB;Dominguez AL;Mirza N;Lin HH;Stein-Streilein J;Gordon S;Lustgarten J
通讯作者：
Lustgarten J
影响因子：
32.4
作者：
Miyao, Takahisa;Floess, Stefan;Hori, Shohei
通讯作者：
Hori, Shohei
DOI：
10.1016/s1387-2656(05)11007-2
发表时间：
2005-01-01
期刊：
BIOTECHNOLOGY ANNUAL REVIEW, VOL 11
影响因子：
--
作者：
Monici, M
通讯作者：
Monici, M
影响因子：
3.7
作者：
Daigneault M;Preston JA;Marriott HM;Whyte MK;Dockrell DH
通讯作者：
Dockrell DH