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流式细胞术检测 CLCF1 蛋白的表达。

Detection of CLCF1 protein expression by flow cytometry.

机构信息

Département de pharmacologie et physiologie, Université de Montréal, Montréal, QC, H3T 1J4, Canada.

Département de microbiologie, infectiologie et immunologie, Université de Montréal, Montréal, QC, H3T 1J4, Canada.

出版信息

Sci Rep. 2024 Jun 10;14(1):13344. doi: 10.1038/s41598-024-64101-9.

DOI:10.1038/s41598-024-64101-9
PMID:38858477
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11164924/
Abstract

Cardiotrophin-like cytokine factor 1 (CLCF1) is an IL-6 family cytokine with neurotrophic and immuno-modulating functions. CLCF1 mRNA has been detected in primary and secondary lymphoid organs, and up-regulation of CLCF1 mRNA levels has been associated with the T helper (Th) 17 polarization. However, information regarding CLCF1 expression by immune cells at the protein level remains scarce. We have developed a methodology that uses a monoclonal antibody (mAb) directed against CLCF1 for the detection of human and mouse CLCF1 by flow cytometry. We have successfully detected CLCF1 protein expression in cells from the mouse pro-B cell line Ba/F3 that were transduced with CLCF1 cDNA. Interestingly, we found that the anti-CLCF1 mAb inhibits CLCF1 biological activity in vitro by binding to an epitope that encompasses site III of the cytokine. Moreover, we have detected CLCF1 expression in mouse splenic T cells, as well as in vitro differentiated Th1 cells. The specificity of the fluorescence signal was demonstrated using Clcf1-deficient lymphocytes generated using a conditional knock-out mouse model. The detection of CLCF1 protein by flow cytometry will be a valuable tool to study CLCF1 expression during normal and pathological immune responses.

摘要

细胞因子样因子 1（CLCF1）是一种具有神经营养和免疫调节功能的白细胞介素 6 家族细胞因子。CLCF1mRNA 已在初级和次级淋巴器官中检测到，并且 CLCF1mRNA 水平的上调与辅助性 T（Th）17 极化有关。然而，关于免疫细胞在蛋白质水平上的 CLCF1 表达的信息仍然很少。我们开发了一种使用针对 CLCF1 的单克隆抗体（mAb）通过流式细胞术检测人源和鼠源 CLCF1 的方法。我们已经成功地检测到转染了 CLCF1 cDNA 的小鼠前 B 细胞系 Ba/F3 细胞中的 CLCF1 蛋白表达。有趣的是，我们发现抗 CLCF1 mAb 通过与细胞因子的 III 位点包含的表位结合来抑制体外 CLCF1 的生物学活性。此外，我们还在小鼠脾 T 细胞以及体外分化的 Th1 细胞中检测到了 CLCF1 的表达。使用条件性敲除小鼠模型生成的 Clcf1 缺陷性淋巴细胞证明了荧光信号的特异性。通过流式细胞术检测 CLCF1 蛋白将成为研究正常和病理免疫反应过程中 CLCF1 表达的有价值的工具。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/677e7c2ef441/41598_2024_64101_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/f77614964017/41598_2024_64101_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/166b6c125825/41598_2024_64101_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/d375c641ad65/41598_2024_64101_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/8811d51edc06/41598_2024_64101_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/677e7c2ef441/41598_2024_64101_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/f77614964017/41598_2024_64101_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/166b6c125825/41598_2024_64101_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/d375c641ad65/41598_2024_64101_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/8811d51edc06/41598_2024_64101_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1d43/11164924/677e7c2ef441/41598_2024_64101_Fig5_HTML.jpg

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