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microfluidic western blot

Zhuoshi Li, Tao Guo, Lei Fang, Nan Li, Xiaochao Wang, Peng Wang, Shilei Zhao, Fengzhou Li, Yanwei Cui, Xin Shu, Lei Zhao, Jinxiu Li, Chundong Gu
Carcinoma‑associated fibroblasts (CAFs) are essential for initiating lung cancer cell invasion and metastasis. An elevated MACC1 expression has been implicated in the progression of lung adenocarcinoma. Hitherto, the role of MACC1 in lung adenocarcinoma‑derived CAFs remains unclear. In this study, CAFs isolated from the tissues of patients with lung adenocarcinoma expressed typical CAF markers (shown by immunohistochemical and immunofluorescence analysis) and exhibited enhanced migration and invasion abilities when co‑cultured with A549 cells in a microfluidic model...
January 30, 2019: International Journal of Oncology
Mona Fani, Majid Rezayi, Zahra Meshkat, Seyed Abdolrahim Rezaee, Manoochehr Makvandi, Ebrahim Abouzari-Lotf, Gordon A Ferns
BACKGROUND: Human T-lymphotropic virus Type 1 (HTLV-1) is a retrovirus that is endemic in some regions of the world. It is known to cause several diseases like adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Serology and molecular methods have been used to detect this virus. Of these, enzyme-linked immunosorbent assay (ELISA) is used as a primary screening method and this is usually followed by western blotting (WB) and polymerase chain reaction (PCR) methods as confirmatory tests...
January 11, 2019: Journal of Cellular Physiology
Xiaolei Yang, Jie Gu, Haichen Lv, Huihua Li, Yunpeng Cheng, Yang Liu, Yinong Jiang
Hyperuricemia is an important risk factor for vascular inflammation, yet the potential mechanisms of uric acid (UA) in endothelial cells are not well understood. UA has been found to stimulate renin-angiotensin system (RAS) activation in human umbilical vein endothelial cells (HUVECs). (Pro)renin receptor ((P)RR) is widely expressed in endothelial cells and able to induce RAS activation. Whether UA-induced endothelial cell inflammation is via up-regulating (P)RR remained unknown. Primary HUVECs were cultured and treated with UA, under the condition of (P)RR or AT1 silencing...
January 2019: Biomedicine & Pharmacotherapy, Biomédecine & Pharmacothérapie
Beiyuan Fan, Xiufeng Li, Lixing Liu, Deyong Chen, Shanshan Cao, Dong Men, Junbo Wang, Jian Chen
Semi-quantitative studies have located varied expressions of β-actin proteins at the population level, questioning their roles as internal controls in western blots, while the absolute copy numbers of β-actins at the single-cell level are missing. In this study, a polymeric microfluidic flow cytometry was used for single-cell analysis, and the absolute copy numbers of single-cell β-actin proteins were quantified as 9.9 ± 4.6 × 10⁵, 6.8 ± 4.0 × 10⁵ and 11.0 ± 5.5 × 10⁵ per cell for A549 (ncell = 14,754), Hep G2 (ncell = 36,949), and HeLa (ncell = 24,383), respectively...
May 22, 2018: Micromachines
Woosuk S Hur, Nima Mazinani, X J David Lu, Leeor S Yefet, James R Byrnes, Laura Ho, Ju Hun Yeon, Sam Filipenko, Alisa S Wolberg, Wilfred A Jefferies, Christian J Kastrup
In cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD), the amyloid β (Aβ) peptide deposits along the vascular lumen, leading to degeneration and dysfunction of surrounding tissues. Activated coagulation factor XIIIa (FXIIIa) covalently cross-links proteins in blood and vasculature, such as in blood clots and on the extracellular matrix. Although FXIIIa co-localizes with Aβ in CAA, the ability of FXIIIa to cross-link Aβ has not been demonstrated. Using Western blotting, kinetic assays, and microfluidic analyses, we show that FXIIIa covalently cross-links Aβ40 into dimers and oligomers ( k cat / Km = 1...
January 11, 2019: Journal of Biological Chemistry
Chao Li, Yi Zhang, Albert M Levin, Bao Yan Fan, Hua Teng, Moleca M Ghannam, Michael Chopp, Zheng Gang Zhang
Proteins and microRNAs (miRNAs) within the axon locally regulate axonal development. However, protein profiles of distal axons of cortical neurons have not been fully investigated. In particular, networks of genes encoding axonal proteins and their related miRNAs in sub compartments of neurons such as axons remain unknown. Using embryonic cortical neurons cultured in a microfluidic device and proteomic approaches, we found that distal axons contain 883 proteins. Bioinformatics analysis revealed that 94 out of these 883 proteins are related to regulating axonal growth...
July 28, 2018: Molecular Neurobiology
Yeong Jun Yu, Young Hye Kim, Kyuhwan Na, Seo Yun Min, Ok Kyung Hwang, Da Kyeong Park, Doo Yeon Kim, Se Hoon Choi, Roger D Kamm, Seok Chung, Jeong Ah Kim
The microfluidic 3D cell culture system has been an attractive model because it mimics the tissue and disease model, thereby expanding our ability to control the local cellular microenvironment. However, these systems still have limited value as quantitative assay tools due to the difficulties associated with the manipulation and maintenance of microfluidic cells, and their lack of compatibility with the high-throughput screening (HTS) analysis system. In this study, we suggest a microchannel-free, 3D cell culture system that has a hydrogel-incorporating unit integrated with a multi-well plate (24- to 96-well plate), which can provide better reproducibility in biological experiments...
August 21, 2018: Lab on a Chip
Zheng Wang, Xiaoju Zhang
In the proteomic and genomic era, lung cancer researchers are increasingly under challenge with traditional protein analyzing tools. High output, multiplexed analytical procedures are in demand for disclosing the post-translational modification, molecular interactions and signaling pathways of proteins precisely, specifically, dynamically and systematically, as well as for identifying novel proteins and their functions. This could be better realized by single-cell proteomic methods than conventional proteomic methods...
2018: Advances in Experimental Medicine and Biology
Chao-Kai Chou, Po-Jung Huang, Pei-Hsiang Tsou, Yongkun Wei, Heng-Huan Lee, Ying-Nai Wang, Yen-Liang Liu, Colin Shi, Hsin-Chih Yeh, Jun Kameoka, Mien-Chie Hung
Protein expression level is critically related to the cell physiological function. However, current methodologies such as Western blot (WB) and Immunohistochemistry (IHC) in analyzing the protein level are rather semi-quantitative and without the information of actual protein concentration. We have developed a microfluidic technique termed a "flow-proteometric platform for analyzing protein concentration (FAP)" that can measure the concentration of a target protein in cells or tissues without the requirement of a calibration standard, e...
October 15, 2018: Biosensors & Bioelectronics
Chi-Chih Kang, Toby M Ward, Jessica Bockhorn, Courtney Schiffman, Haiyan Huang, Mark D Pegram, Amy E Herr
In addition to canonical oncoproteins, truncated isoforms and proteolysis products are implicated in both drug resistance and disease progression. In HER2-positive breast tumors, expression of truncated HER2 isoforms resulting from alternative translation and/or carboxy-terminal fragments (CTFs) resulting from proteolysis (collectively, t-erbB2) have been associated with shortened progression-free survival of patients. Thus, to advance clinical pathology and inform treatment decisions, we developed a high-selectivity cytopathology assay capable of distinguishing t-erbB2 from full-length HER2 expression without the need for isoform-specific antibodies...
2018: NPJ Precision Oncology
Yong-Xin Qin, Zhi-Hui Yang, Xiao-Hui Du, Hui Zhao, Yuan-Bin Liu, Zhe Guo, Qi Wang
Background: The hedgehog signaling system (HHS) plays an important role in the regulation of cell proliferation and differentiation during the embryonic phases. However, little is known about the involvement of HHS in the malignant transformation of cells. This study aimed to detect the role of HHS in the malignant transformation of human bronchial epithelial (16HBE) cells. Methods: In this study, two microfluidic chips were designed to investigate cigarette smoke extract (CSE)-induced malignant transformation of cells...
May 20, 2018: Chinese Medical Journal
M van den Berge, M R Jonker, A Miller-Larsson, D S Postma, I H Heijink
BACKGROUND: COPD patients have increased risk of pneumonia when treated with fluticasone propionate (FP), whereas this is generally not the case with budesonide (BUD) treatment. We hypothesized that BUD and FP differentially affect the expression of immune defense genes. METHODS: Human bronchial epithelial 16HBE cells and air-liquid interface (ALI)-cultured primary bronchial epithelial cells (PBECs) were pre-treated with clinically equipotent concentrations of BUD or FP (0...
June 2018: Pulmonary Pharmacology & Therapeutics
Jiyoung Song, Hyunryul Ryu, Minhwan Chung, Youngtaek Kim, Yannick Blum, Sung Sik Lee, Olivier Pertz, Noo Li Jeon
Recent research on cellular responses is shifting from static observations recorded under static stimuli to real-time monitoring in a dynamic environment. Since cells sense and interact with their surrounding microenvironment, an experimental platform where dynamically changing cellular microenvironments should be recreated in vitro. There has been a lack of microfluidic devices to support spatial and temporal stimulations in a simple and robust manner. Here, we describe a microfluidic device that generates dynamic chemical gradients and pulses in both space and time using a single device...
May 1, 2018: Biosensors & Bioelectronics
Jung-Ming G Lin, Chi-Chih Kang, Yun Zhou, Haiyan Huang, Amy E Herr, Sanjay Kumar
The invasion of malignant cells into tissue is a critical step in the progression of cancer. While it is increasingly appreciated that cells within a tumor differ in their invasive potential, it remains nearly unknown how these differences relate to cell-to-cell variations in protein expression. Here, we introduce a microfluidic platform that integrates measurements of invasive motility and protein expression for single cells, which we use to scrutinize human glioblastoma tumor-initiating cells (TICs). Our live-cell imaging microdevice is comprised of polyacrylamide microchannels that exhibit tissue-like stiffness and present chemokine gradients along each channel...
January 16, 2018: Lab on a Chip
Mohammad A Obeid, Ashref Elburi, Louise C Young, Alexander B Mullen, Rothwelle J Tate, Valerie A Ferro
Small interfering RNAs (siRNA) have a broad potential as therapeutic agents to reversibly silence any target gene of interest. The clinical application of siRNA requires the use of safe and effective delivery systems. In this study, we investigated the use of nonionic surfactant vesicles (NISV) for the delivery of siRNA. Different types of NISV formulations were synthesized by microfluidic mixing and then evaluated for their physiochemical properties and cytotoxicity. The ability of the NISV to carry and transfect siRNA targeting green fluorescent protein (GFP) into A549 that stably express GFP (copGFP-A549) was evaluated...
July 3, 2017: Molecular Pharmaceutics
Axel R Göhring, Stefanie Reuter, Joachim H Clement, Xinlai Cheng, Jannick Theobald, Stefan Wölfl, Ralf Mrowka
PURPOSE: Oct4 was reported to be one of the most important pluripotency transcription factors in the biology of stem cells including cancer stem cells, and progressed malignant cells. Here we report the investigation of gene expression control of Oct4 by selected human microRNAs and the physiological effect of Oct4 silencing in invasive cancer cells. METHODS AND RESULTS: High throughput luciferase activity assay revealed the microRNA-299-3p to be the most effective in reducing gene expression of Oct4, which was confirmed by Western blot analysis and Oct4 promoter activity in a target luciferase assay...
2017: PloS One
Brittany J Sanders, Daniel C Kim, Robert C Dunn
Western blotting is a ubiquitous tool used extensively in the clinical and research settings to identify proteins and characterize their levels. It has rapidly become a mainstay in research laboratories due to its specificity, low cost, and ease of use. The specificity arises from the orthogonal processes used to identify proteins. Samples are first separated based on size and then probed with antibodies specific for the protein of interest. This confirmatory approach helps avoid pitfalls associated with antibody cross-reactivity and specificity issues...
October 21, 2016: Analytical Methods: Advancing Methods and Applications
Wenjun Fan, Jiajun Xie, Jianglong Xia, Yan Zhang, Mengying Yang, Hefei Wang, Yujia Pan, Mengjuan Zhang, Baochun Han, Baitong Wu, Zhijie Hou, Dapeng Liang, Chunli Wang, Jie Xu, Lijuan Song, Quentin Liu
BACKGROUND: The mechanisms of breast cancer collective invasion are poorly understood limiting the metastasis therapy. The ATPase RUVBL1 is frequently overexpressed in various cancers and plays a crucial role in oncogenic process. We further investigated the role of RUVBL1 in promoting collective invasion and uncovered that targeting RUVBL1 could inhibit metastatic progression. METHODS: The expression levels of RUVBL1 and ITFG1 were examined by Western blot and qRT-PCR...
July 2017: Biochimica et biophysica acta. General subjects
Elly Sinkala, Elodie Sollier-Christen, Corinne Renier, Elisabet Rosàs-Canyelles, James Che, Kyra Heirich, Todd A Duncombe, Julea Vlassakis, Kevin A Yamauchi, Haiyan Huang, Stefanie S Jeffrey, Amy E Herr
Circulating tumour cells (CTCs) are rare tumour cells found in the circulatory system of certain cancer patients. The clinical and functional significance of CTCs is still under investigation. Protein profiling of CTCs would complement the recent advances in enumeration, transcriptomic and genomic characterization of these rare cells and help define their characteristics. Here we describe a microfluidic western blot for an eight-plex protein panel for individual CTCs derived from estrogen receptor-positive (ER+) breast cancer patients...
March 23, 2017: Nature Communications
John J Kim, Elly Sinkala, Amy E Herr
Cytology of sparingly available cell samples from both clinical and experimental settings would benefit from high-selectivity protein tools. To minimize cell handling losses in sparse samples, we design a multi-stage assay using a lab-on-a-disc that integrates cell handling and subsequent single-cell western blotting (scWestern). As the two-layer microfluidic device rotates, the induced centrifugal force directs dissociated cells to dams, which in turn localize the cells over microwells. Cells then sediment into the microwells, where the cells are lysed and subjected to scWestern...
February 28, 2017: Lab on a Chip
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