Quantitative Analysis of Immuno-fluorescence of Nuclear Factor-κB Activation_Journal of Biomedical Engineering

Authors：

XIUMin ^1,2 , HEFeng ² , LOUYuanlei ² , XULu ² , XIONGJieqi ² , WANGPing ³ , LIUSisun ⁴ ,  GUOFei ¹

1. Burn Institute, the First Affiliated Hospital of Nanchang University, Nanchang 330006, China;
2. Graduate School, Nanchang University, Nanchang 330031, China;
3. Department of Electronic and Information Engineering, Information Engineering School, Nanchang University, Nanchang 330031, China;
4. Department of Gynecology and Obstetrics, the First Affiliated Hospital of Nanchang University, Nanchang 330006, China;

Corresponding author：

GUOFei, Email: guofei2005@126.com

Keywords：

immuno-fluorescence; fluorescence quantitative analysis; NF-kappa B; human umbilical vein endothelial cells; lipopolysaccharide

DOI：

10.7507/1001-5515.20150121

Video：

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Abstract Full text Figures/Tables Video References Cited by

Immuno-fluorescence technique can qualitatively determine certain nuclear translocation, of which NF-κB/p65 implicates the activation of NF-κB signal pathways. Immuno-fluorescence analysis software with independent property rights is able to quantitatively analyze dynamic location of NF-κB/p65 by computing relative fluorescence units in nuclei and cytoplasm. We verified the quantitative analysis by Western Blot. When we applied the software to analysis of nuclear translocation in lipopolysaccharide (LPS) induced (0.5 h, 1 h, 2 h, 4 h) primary human umbilical vein endothelial cells (HUVECs), we found that nuclear translocation peak showed up at 2h as with calculated Western blot verification results, indicating that the inventive immuno-fluorescence analysis software can be applied to the quantitative analysis of immuno-fluorescence.

Citation： XIUMin, HEFeng, LOUYuanlei, XULu, XIONGJieqi, WANGPing, LIUSisun, GUOFei. Quantitative Analysis of Immuno-fluorescence of Nuclear Factor-κB Activation. Journal of Biomedical Engineering, 2015, 32(3): 669-674. doi: 10.7507/1001-5515.20150121 Copy

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9.	SWEENEY C, LI L, SHANMUGAM R, et al. Nuclear factor-κB is constitutively activated in prostate Cancer in vitro and is overexpressed in prostatic intraepithelial neoplasia and adenocarcinoma of the prostate[J]. Clin Cancer Res, 2004, 10(16):5501-5507.
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11.	朱参胜, 欧阳为明, 刘雪松, 等.抗人TNF单克隆抗体对TNF诱导的NF-κB核转位的抑制作用[J].细胞与分子免疫学杂志, 2004, 20(5):585-587.
12.	刘景臣, 赵立君, 杨小玉, 等.激光共聚焦图像系统与免疫荧光双标记对脊髓缺血再灌注损伤中ICAM-1表达定量分析[J].中国实验诊断学, 2006, 10(9):1036-1038.
13.	NOURSADEGHI M, TSANG J, HAUSTEIN T, et al. Quantitative imaging assay for NF-kB nuclear translocation in primary human macrophages[J]. J Immunol Methods, 2008, 329(1):194-200.
14.	HAYDEN M S, GHOSH S. Shared principles in NF-κB signaling[J]. Cell, 2008, 132(3):344-362.
15.	韩晶, 骞爱荣, 胡丽芳, 等.基于免疫荧光图像的微管蛋白半定量分析[J].第四军医大学学报, 2009(24):2901-2904.
16.	琚玲丽, 刘惠宁.核转录因子κB与恶性肿瘤侵袭转移机制的研究进展[J].中华临床医师杂志:电子版, 2013, 7(1):298-300.

1. TIERI P, TERMANINI A, BELLAVISTA E, et al. Charting the NF-kB pathway interactome map[J]. PLoS One, 2012, 7(3):e32678.
2. ROGERS J A, FUSELER J W. Regulation of NF-kappaB activation and nuclear translocation by exogenous nitric oxide(NO)donors in TNF-alpha activated vascular endothelial cells[J]. Nitric Oxide, 2007, 16(3):379-391.
3. GUZMAN J R, KOO J S, GOLDSMITH J R, et al. Oxymatrine prevents NF-kB nuclear translocation and ameliorates acute intestinal inflammation[J]. Sci Rep, 2013, 3:1629.
4. KADAM S S, TIWARI S, BHONDE R R. Simultaneous isolation of vascular endothelial cells and mesenchymal stem cells from the human umbilical cord[J]. In Vitro Cell Dev Biol Anim, 2009, 45(1/2):23-27.
5. SIOW R C. Culture of human endothelial cells from umbilical veins[J].Methods Mol Biol, 2012, 806:265-274.
6. WANG P, CHEN R, LUO W, et al. Research and implementation of conglutinated macrophage image segmentation based on improved watershed algorithm[J]. Adv Mat Res, 2011, 225:483-487.
7. 王平, 张力, 周长其.基于种子点的粘连巨噬细胞图像的分割方法[J].液晶与显示, 2012, 27(6):808-813.
8. 游迎荣, 范影乐, 庞全.基于距离变换的粘连细胞分割方法[J].计算机工程与应用, 2005, 41(20):206-208.
9. SWEENEY C, LI L, SHANMUGAM R, et al. Nuclear factor-κB is constitutively activated in prostate Cancer in vitro and is overexpressed in prostatic intraepithelial neoplasia and adenocarcinoma of the prostate[J]. Clin Cancer Res, 2004, 10(16):5501-5507.
10. HELLMAN L M, FRIED M G. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions[J]. Nat Protoc, 2007, 2(8):1849-1861.
11. 朱参胜, 欧阳为明, 刘雪松, 等.抗人TNF单克隆抗体对TNF诱导的NF-κB核转位的抑制作用[J].细胞与分子免疫学杂志, 2004, 20(5):585-587.
12. 刘景臣, 赵立君, 杨小玉, 等.激光共聚焦图像系统与免疫荧光双标记对脊髓缺血再灌注损伤中ICAM-1表达定量分析[J].中国实验诊断学, 2006, 10(9):1036-1038.
13. NOURSADEGHI M, TSANG J, HAUSTEIN T, et al. Quantitative imaging assay for NF-kB nuclear translocation in primary human macrophages[J]. J Immunol Methods, 2008, 329(1):194-200.
14. HAYDEN M S, GHOSH S. Shared principles in NF-κB signaling[J]. Cell, 2008, 132(3):344-362.
15. 韩晶, 骞爱荣, 胡丽芳, 等.基于免疫荧光图像的微管蛋白半定量分析[J].第四军医大学学报, 2009(24):2901-2904.
16. 琚玲丽, 刘惠宁.核转录因子κB与恶性肿瘤侵袭转移机制的研究进展[J].中华临床医师杂志:电子版, 2013, 7(1):298-300.

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