期刊论文详细信息
Stem Cell Research
Using a new Lrig1 reporter mouse to assess differences between two Lrig1 antibodies in the intestine - ScienceDirect
Emily J. Poulin1 
关键词: Lrig1;    leucine-rich and immunoglobulin-like domains protein 1;    RFP;    red fluorescent protein;    FACS;    fluorescence-activated cell sorting;    PCR;    polymerase chain reaction;    qRT-PCR;    quantitative real-time PCR;   
DOI  :  10.1016/j.scr.2014.09.002
学科分类:生物科学(综合)
来源: Elsevier
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【 摘 要 】

Lrig1 is an intestinal stem cell marker important for epithelial homeostasis. However, the position of the Lrig1+ population in the intestinal crypt has been debated, largely due to discrepant staining patterns using two Lrig1 antibodies. Here, we set out to decipher the differences between these Lrig1 antibodies to clarify their use for Lrig1-related studies. We confirmed that the commercially available Lrig1-R&D antibody stained the bottom third of the colonic crypt, whereas an independently generated Lrig1-VU antibody recognized a subset of anti-Lrig1-R&D+ cells. Biochemically, we found that anti-Lrig1-VU recognized a non-glycosylated form of Lrig1; in contrast, anti-Lrig1-R&D recognized both glycosylated and non-glycosylated forms of Lrig1. In addition, we generated a reporter mouse (Lrig1-Apple) as an independent readout of Lrig1 transcriptional activity. Flow cytometry of isolated colonic epithelial cells from Lrig1-Apple mice demonstrated anti-Lrig1-R&D recognized mostly RFP-hi cells, while anti-Lrig1-VU recognized cells that were largely RFP-mid. Of note, by qRT-PCR, Lgr5 was expressed in the RFP-hi population, but not in the RFP-mid population. We conclude that anti-Lrig1-R&D appears to recognize all Lrig1+ cells, while anti-Lrig1-VU recognizes a subpopulation of Lrig1+ cells.

【 授权许可】

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