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flow cytometry and compensation

J Hrdý, I Kocourková, R Lodinová-Žádníková, L Kolářová, L Prokešová
Probiotics are believed to prevent or reduce allergy development but the mechanism of their beneficial effect is still poorly understood. Immune characteristics of regulatory T cells (Tregs) in peripheral blood of perinatally probiotic-supplemented children of allergic mothers (51 children), non-supplemented children of allergic mothers (42 children), and non-supplemented children of healthy mothers (28 children) were compared at the age of 6-7 years. A first dose of a probiotic Escherichia coli strain (E. coli O83:K24:H31) was administered within 2 days after the birth and then 12 times during the first months of life and children were followed longitudinally...
September 16, 2016: Beneficial Microbes
Holger Hennig, Paul Rees, Thomas Blasi, Lee Kamentsky, Jane Hung, David Dao, Anne E Carpenter, Andrew Filby
Imaging flow cytometry (IFC) enables the high throughput collection of morphological and spatial information from hundreds of thousands of single cells. This high content, information rich image data can in theory resolve important biological differences among complex, often heterogeneous biological samples. However, data analysis is often performed in a highly manual and subjective manner using very limited image analysis techniques in combination with conventional flow cytometry gating strategies. This approach is not scalable to the hundreds of available image-based features per cell and thus makes use of only a fraction of the spatial and morphometric information...
September 1, 2016: Methods: a Companion to Methods in Enzymology
Sandrine Schmutz, Mariana Valente, Ana Cumano, Sophie Novault
Flow cytometry, initially developed to analyze surface protein expression in hematopoietic cells, has increased in analytical complexity and is now widely used to identify cells from different tissues and organisms. As a consequence, data analysis became increasingly difficult due the need of large multi-parametric compensation matrices and to the eventual auto-fluorescence frequently found in cell suspensions obtained from solid organs. In contrast with conventional flow cytometry that detects the emission peak of fluorochromes, spectral flow cytometry distinguishes the shapes of emission spectra along a large range of continuous wave lengths...
2016: PloS One
Xuesong Deng, Tongming Deng, Yong Ni, Yongqiang Zhan, Wenlong Huang, Jianfeng Liu, Caixian Liao
It has been observed that polymorphonuclear neutrophils (PMN) increase in number and function during obstructive jaundice (OJ). However, the precise mechanisms underlying PMN apoptosis during OJ remain poorly understood. The aim of the present study was to investigate the modulation of cytochrome c (Cytc) on the mitochondrial signaling pathway in bile duct-ligated (BDL) rats and the effect on PMN apoptosis following the intravenous administration of Cytc. Rats were randomly divided into four groups: A control group, a sham group, a BDL group and a BDL + Cytc group (rats with common bile duct ligation as well as Cytc intravenous injection)...
July 2016: Experimental and Therapeutic Medicine
Victoria L Gadd, Preya J Patel, Sara Jose, Leigh Horsfall, Elizabeth E Powell, Katharine M Irvine
BACKGROUND AND AIMS: Liver and systemic inflammatory factors influence monocyte phenotype and function, which has implications for hepatic recruitment and subsequent inflammatory and fibrogenic responses, as well as host defence. METHODS: Peripheral blood monocyte surface marker (CD14, CD16, CD163, CSF1R, CCR2, CCR4, CCR5, CXCR3, CXCR4, CX3CR1, HLA-DR, CD62L, SIGLEC-1) expression and capacity for phagocytosis, oxidative burst and LPS-stimulated TNF production were assessed in patients with hepatitis C (HCV) (n = 39) or non-alcoholic fatty liver disease (NAFLD) (n = 34) (classified as non-advanced disease, compensated cirrhosis and decompensated cirrhosis) and healthy controls (n = 11) by flow cytometry...
2016: PloS One
Qiao-rong Huang, Bo Wen, Xue Li, Lian Hu, Xue-mei Wan, Sheng-ping Zeng, Wen-tong Meng
OBJECTIVE: To establish an assay using 9-color flow cytometry immunophenotyping to detect activation and apoptosis of human TCR Vβ lymphocyte subpopulations in peripheral blood samples. METHODS: We used 5 antibodies (CD3, CD4, CD8, CD95, CD69), phospholipids binding proteins Annexin V, TCR Vβ Repertoire Kit and nucleus dye DAPI to establish a 9-color flow cytometry assay. Peripheral blood samples were taken from eight healthy people for test of antibodies and determination of optimal PMT and staining method (single-stained vs stained with all but one antibody)...
March 2016: Sichuan da Xue Xue Bao. Yi Xue Ban, Journal of Sichuan University. Medical Science Edition
Juan M Agraz-Cibrian, Jorge E Segura-Ortega, Vidal Delgado-Rizo, Mary Fafutis-Morris
INTRODUCTION: Liver cirrhosis (LC) constitutes one of the main 10 causes of death worldwide. LC has a characteristic asymptomatic compensated phase followed by a progressive decompensated phase, in which diverse complications are presented. LC patients are highly prone to bacterial infections. The neutrophils in these patients present defects in the production of oxygen radicals, which are essential for bacteria elimination as in the activation of neutrophil extracellular traps (NETs)...
2016: Journal of Infection in Developing Countries
Oriol Juanola, Isabel Gómez-Hurtado, Pedro Zapater, Alba Moratalla, Esther Caparrós, Paula Piñero, José M González-Navajas, Paula Giménez, José Such, Rubén Francés
BACKGROUND&AIMS: Norfloxacin exerts immunomodulatory effects in cirrhosis beyond its bactericidal activity. We aimed at identifying the role of regulatory T (Treg) cells in the norfloxacin mechanism that compensates the inflammatory environment in cirrhosis. PATIENTS&METHODS: Consecutively admitted patients with cirrhosis and ascitic fluid (AF) with: spontaneous bacterial peritonitis (SBP), non-infected AF, and norfloxacin as secondary SBP prophylaxis (SID group)...
May 23, 2016: Liver International: Official Journal of the International Association for the Study of the Liver
Xiao-Xi Kan, Qi Li, Xi Chen, Ya-Jie Wang, Yu-Jie Li, Qing Yang, Hong-Bin Xiao, Zhi-Xin Wang, Ying Chen, Xiao-Gang Weng, Wei-Yan Cai, Xiao-Xin Zhu
Currently, liver cancer is the sixth most prevalent cancer and the third most common cause of cancer-related death. However, effective chemotherapeutic drugs with low drug resistance and few side-effects for the clinical treatment of liver cancer are lacking. Therefore, the search for novel drugs to compensate for the defects of existing drugs is urgently needed. Herein, we successfully screened an extract named from Stellera chamaejasme L. (SCL), a historically confimed antitumor plant, through a novel extraction platform...
June 2016: Oncology Reports
Kathryn Fuller, Matthew D Linden, Tracey Lee-Pullen, Clayton Fragall, Wendy N Erber, Kimberley J Röhrig
Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype...
June 2016: Advances in Physiology Education
Quanwen Li, Nardhy Gomez-Lopez, Sascha Drewlo, Elly Sanchez-Rodriguez, Jing Dai, Elizabeth E Puscheck, Daniel A Rappolee
Assays for embryonic stem cells (ESCs) of the blastocyst are needed to quantify stress-induced decreases of potent subpopulations. High-throughput screens (HTSs) of stressed ESCs quantify embryonic stress, diminishing laboratory animal needs. Normal or stress-induced ESC differentiation is marked by Rex1 potency factor loss. Potency reporter ESC assays were developed, using low-stress techniques to create transgenic ESCs. Rex1 and Oct4 promoters drove RFP and green fluorescent protein (GFP) expression, respectively...
February 15, 2016: Stem Cells and Development
Peter T White, Chitra Subramanian, Qing Zhu, Huaping Zhang, Huiping Zhao, Robert Gallagher, Barbara N Timmermann, Brian S J Blagg, Mark S Cohen
BACKGROUND: Thyroid cancer stem cells (CSCs) with ALDH and CD44 markers contribute to tumor growth and aggressiveness. We hypothesized that novel HSP90 inhibitors (KU711, WGA-TA) and 17-AAG can effectively target the function of thyroid CSCs in vitro and prevent migration and invasion. METHODS: Validated papillary (TPC1), follicular (FTC238,WRO), and anaplastic (ACT1) human thyroid cancer cell lines were treated with 3 HSP90 inhibitors. CSCs were quantified for aldehyde dehydrogenase by flow cytometry, CD44 expression by Western blot, and thyrosphere formation assay...
January 2016: Surgery
Yang Peng, Yuxiang Dong, Ram I Mahato
Mycophenolic acid (MPA) is one of the most commonly used immunosuppressive drugs for improving the outcome of cell and organ transplantations. However, an undesired adverse effect of MPA impedes its application in the clinics for post-transplant patients. By conjugating MPA to quinic acid (QA) via amide bonds, we synthesized a novel immunosuppressant, N-[2-[[(4E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-1-oxo-4-hexen-1-yl]amino]ethyl]-(1α,3R,4α,5R)-1,3,4,5-tetrakis(acetyloxy)cyclohexanecarboxamide (abbreviated as MQ4), which exhibits improved stability demonstrated by its incubation in vitro with human plasma, suggesting its better resistance to hydrolytic degradation induced by plasma enzyme...
December 7, 2015: Molecular Pharmaceutics
A Graham Pockley, Gemma A Foulds, Julie A Oughton, Nancy I Kerkvliet, Gabriele Multhoff
Fluorescent immunophenotyping uses fluorescently-conjugated antibodies to identify, characterize and quantify distinct subpopulations of cells within heterogeneous single-cell populations, either in the context of tissue (using fluorescence and imaging microscopy) or in a single-cell suspension (using multiparameter imaging microscopy, imaging cytometry, and/or flow cytometry). Flow cytometry is an optical, laser-based technology which analyzes the physical and fluorescent properties of cells in suspension in real-time as they flow through the instrument...
2015: Current Protocols in Toxicology
Gábor Szalóki, Katalin Goda
No abstract text is available yet for this article.
November 2015: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Inna Vainshtein, Amy K Schneider, Bo Sun, Martin Schwickart, Lorin K Roskos, Meina Liang
BACKGROUND: Receptor occupancy (RO) assays measure drug target engagement, and are used as pharmacodynamic (PD) biomarkers. RO assays are commonly performed by flow cytometry and often require multiplexing for assessment of multiple PD biomarkers when specimen volumes are limited. We present multiplexed RO assays for an IGF1R-EGFR bispecific antibody (Bs-Ab) and a CTLA4-Ig recombinant fusion protein to demonstrate key considerations for accurate RO assessment. METHODS: RO in cynomolgus monkeys was determined in whole blood using flow cytometry...
March 2016: Cytometry. Part B, Clinical Cytometry
Sarah Lieder, Michael Jahn, Joachim Koepff, Susann Müller, Ralf Takors
Cellular response to different types of stress is the hallmark of the cell's strategy for survival. How organisms adjust their cell cycle dynamics to compensate for changes in environmental conditions is an important unanswered question in bacterial physiology. A cell using binary fission for reproduction passes through three stages during its cell cycle: a stage from cell birth to initiation of replication, a DNA replication phase and a period of cell division. We present a detailed analysis of durations of cell cycle phases, investigating their dynamics under environmental stress conditions...
January 2016: Biotechnology Journal
Matthew R DiStasi, Julie A Mund, H Glenn Bohlen, Steven J Miller, David A Ingram, Michael C Dalsing, Joseph L Unthank
The present study was undertaken to establish the role of NADPH oxidase (Nox) in impaired vascular compensation to arterial occlusion that occurs in the presence of risk factors associated with oxidative stress. Diet-induced obese (DIO) mice characterized by multiple comorbidities including diabetes and hyperlipidemia were used as a preclinical model. Arterial occlusion was induced by distal femoral artery ligation in lean and DIO mice. Proximal collateral arteries were identified as the site of major (∼70%) vascular resistance to calf perfusion by distal arterial pressures, which decreased from ∼80 to ∼30 mmHg with ligation in both lean and DIO mice...
October 2015: American Journal of Physiology. Heart and Circulatory Physiology
Tiara Byrd, Karen D Carr, John C Norman, Leslie Huye, Meenakshi Hegde, Nabil Ahmed
Compensation is a critical process for the unbiased analysis of flow cytometry data. Numerous compensation strategies exist, including the use of bead-based products. The purpose of this study was to determine whether beads, specifically polystyrene microspheres (PSMS) compare to the use of primary leukocytes for single color based compensation when conducting polychromatic flow cytometry. To do so, we stained individual tubes of both PSMS and leukocytes with panel specific antibodies conjugated to fluorochromes corresponding to fluorescent channels FL1-FL10...
November 2015: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Sheng-Huei Yang, Hung-Yun Lin, Vincent H S Chang, Chien-Chung Chen, Yun-Ru Liu, Jinghan Wang, Keqiang Zhang, Xiaoqing Jiang, Yun Yen
Gefitinib resistance has been shown to complicate cancer therapy. Lovastatin is a proteasome inhibitor that enhances gefitinib-induced antiproliferation in non-small cell lung cancer. The objective of this study is to investigate the mechanism of lovastatin-induced antiproliferation in gefitinib-resistant human cholangiocarcinoma.Two gefitinib-resistant cholangiocarcinoma cell lines, SSP-25 and HuH-28, were used in this study to determine how to compensate gefitinib resistance. The combined effect of these two drugs was examined using the MTT assay, qPCR, immunoblotting, flow cytometry, and in vivo xenograft...
September 15, 2015: Oncotarget
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