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Intracellular Flow Cytometry

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https://www.readbyqxmd.com/read/28224564/increased-glucocorticoid-receptor-expression-in-sepsis-is-related-to-heat-shock-proteins-cytokines-and-cortisol-and-is-associated-with-increased-mortality
#1
Konstantinos Vardas, Stavroula Ilia, Amalia Sertedaki, Evangelia Charmandari, Efrossini Briassouli, Dimitris Goukos, Kleovoulos Apostolou, Katerina Psarra, Efthimia Botoula, Stylianos Tsagarakis, Eleni Magira, Christina Routsi, Constantine A Stratakis, Serafim Nanas, George Briassoulis
BACKGROUND: The purposes of this study are to examine if the human glucocorticoid receptor (hGR) isoform-α mRNA and hGR protein expressions are deficient in the acute phase of sepsis (S) compared to systemic inflammatory response syndrome (SIRS) and healthy subjects (H) and to evaluate if the hGRα and hGR alterations are associated with cortisol changes and if they are related to (1) extracellular and intracellular heat shock proteins (HSP) 72 and 90α; (2) ACTH, prolactin, and interleukins (ILs); and (3) outcome...
December 2017: Intensive Care Medicine Experimental
https://www.readbyqxmd.com/read/28224106/impact-of-high-dose-vitamin-c-on-platelet-function
#2
Bassem M Mohammed, Kimberly W Sanford, Bernard J Fisher, Erika J Martin, Daniel Contaifer, Urszula Osinska Warncke, Dayanjan S Wijesinghe, Charles E Chalfant, Donald F Brophy, Alpha A Fowler Iii, Ramesh Natarajan
AIM: To examine the effect of high doses of vitamin C (VitC) on ex vivo human platelets (PLTs). METHODS: Platelet concentrates collected for therapeutic or prophylactic transfusions were exposed to: (1) normal saline (control); (2) 0.3 mmol/L VitC (Lo VitC); or (3) 3 mmol/L VitC (Hi VitC, final concentrations) and stored appropriately. The VitC additive was preservative-free buffered ascorbic acid in water, pH 5.5 to 7.0, adjusted with sodium bicarbonate and sodium hydroxide...
February 4, 2017: World Journal of Critical Care Medicine
https://www.readbyqxmd.com/read/28223800/comparison-of-the-cellular-transport-mechanism-of-cationic-star-shaped-polymers-and-liposomes-in-hacat-cells
#3
Heng-Cong Luo, Na Li, Li Yan, Kai-Jin Mai, Kan Sun, Wei Wang, Guo-Juan Lao, Chuan Yang, Li-Ming Zhang, Meng Ren
Several biological barriers must be overcome to achieve efficient nonviral gene delivery. These barriers include target cell uptake, lysosomal degradation, and dissociation from the carrier. In this study, we compared the differences in the uptake mechanism of cationic, star-shaped polymer/MMP-9siRNA complexes (β-CD-(D3)7/MMP-9siRNA complexes: polyplexes) and commercial liposome/MMP-9siRNA complexes (Lipofectamine(®) 2000/MMP-9siRNA complexes: liposomes). The uptake pathway and transfection efficiency of the polyplexes and liposomes were determined by fluorescence microscopy, flow cytometry, and reverse transcriptase-polymerase chain reaction...
2017: International Journal of Nanomedicine
https://www.readbyqxmd.com/read/28218958/cd137-is-a-useful-marker-for-identifying-cd4-t-cell-responses-to-mycobacterium-tuberculosis
#4
Zhuo-Hong Yan, Xiao-Fang Zheng, Ling Yi, Jing Wang, Xiao-Jue Wang, Pan-Jian Wei, Hong-Yan Jia, Li-Jun Zhou, Yan-Lin Zhao, Hong-Tao Zhang
Upregulation of CD137 on recently activated CD8(+) T cells has been used to identify rare viral and tumor antigen-specific T cells from the peripheral blood. We aimed to evaluate the accuracy of CD137 for identifying Mycobacterium tuberculosis (Mtb)-reactive CD4(+) T cells in the peripheral blood of infected individuals by flow cytometry and to investigate the characteristics of these CD137(+) CD4(+) T cells. We initially enrolled 31 active tuberculosis (TB) patients, 31 individuals with latent TB infection (LTBI), and 25 healthy donors...
February 20, 2017: Scandinavian Journal of Immunology
https://www.readbyqxmd.com/read/28218221/gene-expressions-underlying-mishandled-calcium-clearance-and-elevated-generation-of-reactive-oxygen-species-in-the-coronary-artery-smooth-muscle-cells-of-chronic-heart-failure-rats
#5
Liang Ding, Xian-Xiu Su, Wen-Hui Zhang, Yu-Xiang Xu, Xue-Feng Pan
BACKGROUND: The calcium clearance and reactive oxygen species (ROS) generations in the coronary artery smooth muscle cells in chronic heart failure (HF) have not been fully investigated. Therefore, we attempted to understand the gene expressions underlying the mishandling of calcium clearance and the accumulations of ROS. METHODS: We initially established an animal model of chronic HF by making the left anterior descending coronary artery ligation (CAL) in rats, and then isolated the coronary artery vascular smooth muscle cells from the ischemic and the nonischemic parts of the coronary artery vessels in 12 weeks after CAL operation...
2017: Chinese Medical Journal
https://www.readbyqxmd.com/read/28217898/metal-induced-changes-in-trivalent-chromium-resistant-alcaligenes-faecalis-vitsim2
#6
Shiny C Matilda, Chittibabu Shanthi
The changes induced in bacterial strains under stress conditions provide an insight into metal resistance strategies. Trivalent chromium resistant bacterium were isolated and identified by 16S rRNA gene sequencing and designated as Alcaligenes faecalis VITSIM2. The growth pattern was monitored. The organism also showed resistance to copper, cadmium, and certain antibiotics. The differentially expressed proteins in SDS PAGE were identified by mass spectrometry as flagellin and 50S ribosomal L36 protein. The morphological changes were identified by scanning electron microscopy...
February 20, 2017: Journal of Basic Microbiology
https://www.readbyqxmd.com/read/28216965/mutational-analysis-of-hepatitis-e-virus-orf1-y-domain-effects-on-rna-replication-and-virion-infectivity
#7
Mohammad Khalid Parvez
AIM: To investigate the role of non-structural open reading frame 1 "Y-domain" sequences in the hepatitis E virus (HEV) life cycle. METHODS: Sequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry...
January 28, 2017: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/28214951/inhibition-of-jak-stat-pathway-restrains-tslp-activated-dendritic-cells-mediated-inflammatory-t-helper-type-2-cell-response-in-allergic-rhinitis
#8
Zhaohui Shi, Weihong Jiang, Min Wang, Xiaocheng Wang, Xiaoyuan Li, Xiaodong Chen, Li Qiao
Thymic stromal lymphopoietin (TSLP) has recently been implicated as a key molecule for initiating allergic rhinitis (AR) at the cell-dendritic cell (DC) interface. Previous studies demonstrated that TSLP activated DCs to express more OX40 ligand (OX40L), which is associated with the initiation of T helper type 2 (Th2) cell responses. STAT phosphorylation has been reported to be promoted by TSLP. Thus, we investigated if the JAK/STAT pathway inhibitor CYT387 could affect TSLP-DC-mediated Th2 cell response in naive T cell and AR mice model...
February 18, 2017: Molecular and Cellular Biochemistry
https://www.readbyqxmd.com/read/28214891/up-regulated-atf4-expression-increases-cell-sensitivity-to-apoptosis-in-response-to-radiation
#9
Ying Zong, Shijie Feng, Jinwei Cheng, Chenlin Yu, Guocai Lu
BACKGROUND/AIMS: Activating transcription factor 4 (ATF4) is a member of the activating transcription factor family which regulates the expression of genes involved in amino acid metabolism, redox homeostasis and ER stress responses. ATF4 is also over-expressed in human solid tumors, although its effect on responsiveness to radiation is largely unexplored. METHODS: Real-time PCR was used to detect ATF4 mRNA levels in cells treated with different doses of 60Coγ radiation...
February 13, 2017: Cellular Physiology and Biochemistry
https://www.readbyqxmd.com/read/28205169/quantitative-measurement-of-cholesterol-in-cell-populations-using-flow-cytometry-and-fluorescent-perfringolysin-o
#10
Jian Li, Peter L Lee, Suzanne R Pfeffer
Methods to quantify intracellular cholesterol are valuable for the study of its trafficking and storage in normal cells and in lysosomal storage disorders. Traditionally, cholesterol has been tracked using the small molecule, filipin. Filipin can be difficult to visualize and visualization can be cytotoxic as it requires UV illumination. Here we describe a method to measure cholesterol using a fluorescently labeled, mutant form of Perfringolysin O, a soluble protein toxin that binds cholesterol specifically...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28204825/morroniside-protects-sk-n-sh-human-neuroblastoma-cells-against-h2o2-induced-damage
#11
Jing-Xing Zhang, Rui Wang, Jin Xi, Lin Shen, An-You Zhu, Qi Qi, Qi-Yi Wang, Lun-Jun Zhang, Feng-Chao Wang, He-Zuo Lü, Jian-Guo Hu
Oxidative stress-induced cell injury has been linked to the pathogenesis of neurodegenerative disorders such as spinal cord injury, Parkinson's disease, and multiple sclerosis. Morroniside is an antioxidant derived from the Chinese herb Shan-Zhu-Yu. The present study investigated the neuroprotective effect of morroniside against hydrogen peroxide (H2O2)-induced cell death in SK-N-SH human neuroblastoma cells. H2O2 increased cell apoptosis, as determined by flow cytometry and Hoechst 33342 staining. This effect was reversed by pretreatment with morroniside at concentrations of 1-100 µM...
March 2017: International Journal of Molecular Medicine
https://www.readbyqxmd.com/read/28203317/synthetic-isoliquiritigenin-inhibits-human-tongue-squamous-carcinoma-cells-through-its-antioxidant-mechanism
#12
Cuilan Hou, Wenguang Li, Zengyou Li, Jing Gao, Zhenjie Chen, Xiqiong Zhao, Yaya Yang, Xiaoyu Zhang, Yong Song
Isoliquiritigenin (ISL), a natural antioxidant, has antitumor activity in different types of cancer cells. However the antitumor effect of ISL on human tongue squamous carcinoma cells (TSCC) is not clear. Here we aimed to investigate the effects of synthetic isoliquiritigenin (S-ISL) on TSCC and elucidate the underlying mechanisms. S-ISL was synthesized and elucidated from its nuclear magnetic resonance spectrum and examined using high performance liquid chromatography. The effects of S-ISL on TSCC cells (Tca8113) were evaluated in relation to cell proliferation, apoptosis and adhesion, migration, and invasion using sulforhodamine B assay, fluorescence microscopy technique, flow cytometry (FCM) analysis, and Boyden chamber assay...
2017: Oxidative Medicine and Cellular Longevity
https://www.readbyqxmd.com/read/28202745/plasma-levels-of-eicosapentaenoic-acid-are-associated-with-anti-tnf-responsiveness-in-rheumatoid-arthritis-and-inhibit-the-etanercept-driven-rise-in-th17-cell-differentiation-in-vitro
#13
Louisa Jeffery, Helena L Fisk, Philip C Calder, Andrew Filer, Karim Raza, Christopher D Buckley, Iain McInnes, Peter C Taylor, Benjamin A Fisher
OBJECTIVE: To determine whether levels of plasma n-3 polyunsaturated fatty acids are associated with response to antitumor necrosis factor (anti-TNF) agents in rheumatoid arthritis (RA), and whether this putative effect may have its basis in altering anti-TNF-driven Th17 cell differentiation. METHODS: Plasma was collected at baseline and after 3 months of anti-TNF treatment in 22 patients with established RA, and fatty acid composition of the phosphatidylcholine (PC) component was measured...
February 15, 2017: Journal of Rheumatology
https://www.readbyqxmd.com/read/28202418/ros-txnip-pathway-contributes-to-thrombin-induced-nlrp3-inflammasome-activation-and-cell-apoptosis-in-bv2-cells
#14
Dandan Zuo, Xinchun Ye, Lu Yu, Liang Zhang, Jiao Tang, Chengcheng Cui, Lei Bao, Kun Zan, Zuohui Zhang, Xinxin Yang, Hao Chen, Hai Tang, Jie Zu, Hongjuan Shi, Guiyun Cui
There is no effective therapy for intracerebral hemorrhage (ICH) because of poor understanding of the mechanisms of brain injury after hemorrhage. The NLRP3 inflammasome, as a vital component of innate immune system, which is associated with a wide range of human CNS disorders, including ICH. But its detailed mechanisms in ICH remain mainly unclear. In this study, BV2 cells with thrombin exposure were used to investigate the role of NLRP3 inflammasome in thrombin-induced brain injury. We used western blot to detect NLRP3 inflammasome activation and the expression of thioredoxin binding protein (TXNIP), DCFH-DA to investigate intracellular reactive oxygen species (ROS), flow cytometry to analyze apoptosis...
February 12, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28202394/platelet-rich-plasma-prp-induces-chondroprotection-via-increasing-autophagy-anti-inflammatory-markers-and-decreasing-apoptosis-in-human-osteoarthritic-cartilage
#15
Mayssam Moussa, Daniel Lajeunesse, George Hilal, Oula El Atat, Gaby Haykal, Rim Serhal, Antonio Chalhoub, Charbel Khalil, Nada Alaaeddine
OBJECTIVES: Autophagy constitutes a defense mechanism to overcome aging and apoptosis in osteoarthritic cartilage. Several cytokines and transcription factors are linked to autophagy and play an important role in the degradative cascade in osteoarthritis (OA). Cell therapy such as platelet rich plasma (PRP) has recently emerged as a promising therapeutic tool for many diseases including OA. However, its mechanism of action on improving cartilage repair remains to be determined. The purpose of this study is to investigate the effect of PRP on osteoarthritic chondrocytes and to elucidate the mechanism by which PRP contributes to cartilage regeneration...
February 13, 2017: Experimental Cell Research
https://www.readbyqxmd.com/read/28202289/rolipram-potentiates-bevacizumab-induced-cell-death-in-human-glioblastoma-stem-like-cells
#16
Sara Ramezani, Nasim Vousooghi, Fatemeh Ramezani Kapourchali, Mahmoudreza Hadjighasem, Parisa Hayat, Naser Amini, Mohammad Taghi Joghataei
AIMS: Glioblastoma cancer stem-like cells (GCSCs) promote themselves proliferation by secreting the vascular endothelial growth factor A (VEGFA) in an autocrine manner, positively regulated by phosphodiesterase IV (PDE4). In the current study, we investigated the putative cytotoxic effect of bevacizumab, a VEGFA blocker, alone and in combination with a specific inhibitor of PDE4 called rolipram on GCSCs isolated from human surgical tumor specimen with a focus on PI3K/AKT pathway. MAIN METHODS: CD133+/CD15+ GCSCs were characterized by flow cytometry and expanded in a serum-free primary culture system...
February 12, 2017: Life Sciences
https://www.readbyqxmd.com/read/28197337/defective-t-cell-control-of-epstein-barr-virus-infection-in-multiple-sclerosis
#17
Michael P Pender, Peter A Csurhes, Jacqueline M Burrows, Scott R Burrows
Mounting evidence indicates that infection with Epstein-Barr virus (EBV) has a major role in the pathogenesis of multiple sclerosis (MS). Defective elimination of EBV-infected B cells by CD8(+) T cells might cause MS by allowing EBV-infected autoreactive B cells to accumulate in the brain. Here we undertake a comprehensive analysis of the T-cell response to EBV in MS, using flow cytometry and intracellular IFN-γ staining to measure T-cell responses to EBV-infected autologous lymphoblastoid cell lines and pools of human leukocyte antigen (HLA)-class-I-restricted peptides from EBV lytic or latent proteins and cytomegalovirus (CMV), in 95 patients and 56 EBV-seropositive healthy subjects...
January 2017: Clinical & Translational Immunology
https://www.readbyqxmd.com/read/28193524/synthetic-peptide-ala-arg-glu-gly-glu-met-abolishes-pro-proliferative-and-anti-apoptotic-effects-of-high-glucose-in-vascular-smooth-muscle-cells
#18
Xiaozhou Cao, Yi Lyu, Junyu Ning, Xiaozhi Tang, Xinchun Shen
Apoptosis plays a critical role in normal vascular development and atherosclerosis. However, high glucose has been reported to generate a certain level of ROS that can inhibit vascular smooth muscle cell (VSMC) apoptosis, with the underlying mechanism remaining unclear. In this study, a synthetic peptide AREGEM (Ala-Arg-Glu-Gly-Glu-Met) exhibited antioxidative effects and was used to investigate its function in VSMCs during hyperglycaemia. MTT assay results demonstrated that AREGEM significantly attenuated high glucose-induced VSMCs proliferation...
February 11, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28192188/hepatitis-c-virus-induced-nk-cell-activation-causes-metzincin-mediated-cd16-cleavage-and-impaired-antibody-dependent-cytotoxicity
#19
Barbara Oliviero, Stefania Mantovani, Stefania Varchetta, Dalila Mele, Giulia Grossi, Serena Ludovisi, Elisa Nuti, Armando Rossello, Mario U Mondelli
BACKGROUND & AIMS: The Fc receptor family for IgG type III (FcγRIII, CD16) is an activating receptor on natural killer (NK) cells and an essential mediator of antibody-dependent cellular cytotoxicity (ADCC). There is only limited information on its role during chronic hepatitis C virus (HCV) infection. We studied CD16 expression in relation to NK cell functional activity in HCV-infected patients and sought mechanistic insights into virus-induced modulation. METHODS: NK cell CD16 expression and activation status were evaluated ex vivo by flow cytometry in HCV-infected patients and healthy controls (HC) as well as in vitro after co-culture with HCV-infected Huh 7...
February 9, 2017: Journal of Hepatology
https://www.readbyqxmd.com/read/28187381/inherently-fluorescent-polystyrene-microspheres-for-coating-sensing-and-cellular-imaging
#20
Jian-Bo Qu, Yu-Liang Xu, Yu Liu, Yanan Wang, Yuanhong Sui, Jian-Guo Liu, Xiaojuan Wang
Commercially available polystyrene (PS) fluorescent microspheres are widely used in biological field for tracing, in vivo imaging and calibration of flow cytometry, among other applications. However, these particles do suffer from some drawbacks such as the leakage and photobleaching of organic dyes within them. In the present study, inherently fluorescent properties of PS based microspheres have been explored for the first time. Here we find that a simple chloromethylation reaction endows the polystyrene particles with inherent fluorescence without any subsequent conjugation of an external fluorophore...
February 4, 2017: Colloids and Surfaces. B, Biointerfaces
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