scholarly journals Analysis of in vitro lymphocyte adhesion and transendothelial migration by fluorescent-beads-based flow cytometric cell counting

Cytometry ◽  
1998 ◽  
Vol 32 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Grietje Molema ◽  
Geert Mesander ◽  
Bart Jan Kroesen ◽  
Wijnand Helfrich ◽  
Dirk K.F. Meijer ◽  
...  
Keyword(s):  
Transendothelial Migration ◽  
Cell Counting ◽  
Lymphocyte Adhesion ◽  
Flow Cytometric ◽  
Fluorescent Beads ◽  
Flow Cytometric Cell

Flow-cytometric cell counting and DNA estimation for the study of plant cell population dynamics

1994 ◽  
Vol 39 (3) ◽  
pp. 251-259 ◽  
Author(s):  
Fernando T. Nicoloso ◽  
John Val ◽  
Maarten van der Keur ◽  
Frank van Iren ◽  
Jan W. Kijne
Keyword(s):  
Population Dynamics ◽  
Cell Population ◽  
Plant Cell ◽  
Cell Counting ◽  
Cell Population Dynamics ◽  
Flow Cytometric ◽  
Flow Cytometric Cell

Establishment of Clonal Colony-Forming Assay System for Pancreatic Stem/Progenitor Cells

2002 ◽  
Vol 11 (5) ◽  
pp. 451-453 ◽  
Author(s):  
Atsushi Suzuki ◽  
Kazunobu Oyama ◽  
Katashi Fukao ◽  
Hiromitsu Nakauchi ◽  
Hideki Taniguchi
Keyword(s):  
Progenitor Cells ◽  
Assay System ◽  
Cell Potential ◽  
Differentiated Cells ◽  
Flow Cytometric ◽  
Stem Cell Potential ◽  
Colony Forming Assay ◽  
Flow Cytometric Cell ◽  
Clonal Assay

Pluripotent stem cells found in a number of organs are usually in small cell populations. However, under adaptive stimulation, they enter the stage of growth and differentiation to compensate for the loss of differentiated cells. To analyze stem cell potential precisely, the exclusion of other differentiated cells and a clonal assay system are strongly required. In this study, we established a colony-forming assay system for pancreatic stem/progenitor cells in vitro. In this culture condition, they received signals for growth and differentiation, and formed clonal colonies including pancreatic endocrine-lineage cells, such as α and β cells. By combining this culture system with flow cytometric cell sorting, pancreatic stem/progenitor cells will be enriched, and their potential can be analyzed precisely in single cell-based experiments.

Bead-Based Flow-Cytometric Cell Counting of Live and Dead Bacteria

2019 ◽  
pp. 123-134 ◽  
Author(s):  
Fang Ou ◽  
Cushla McGoverin ◽  
Joni White ◽  
Simon Swift ◽  
Frédérique Vanholsbeeck
Keyword(s):  
Cell Counting ◽  
Flow Cytometric ◽  
Flow Cytometric Cell

Proteomics-Based Characterization of the Effects of MMP14 on the Protein Content of Exosomes from Corneal Fibroblasts

2020 ◽  
Vol 27 (10) ◽  
pp. 979-988
Author(s):  
Kyu-Yeon Han ◽  
Jin-Hong Chang ◽  
Dimitri T. Azar
Keyword(s):  
Protein Content ◽  
Catalytic Domain ◽  
Protein Composition ◽  
Proteomics Analysis ◽  
Knock Out ◽  
Corneal Fibroblast ◽  
Flow Cytometric ◽  
Corneal Fibroblasts ◽  
Liquid Chromatography Tandem Mass

Background: Exosomes secreted by corneal fibroblasts contain matrix metalloproteinase (MMP) 14, which is known to influence pro-MMP2 accumulation on exosomes. Accordingly, we hypothesized that the enzymatic activity of MMP14 may alter the protein content of corneal fibroblast- secreted exosomes. Objective: The aim of this study was to investigate the effects of MMP14 on the composition and biological activity of corneal fibroblast-derived exosomes. Methods: Knock out of the catalytic domain (ΔExon4) of MMP14 in corneal fibroblasts was used to determine the effect of MMP14 expression on the characteristics of fibroblast-secreted exosomes. The amount of secreted proteins and their size distribution were measured using Nano Tracking Analysis. Proteins within exosomes from wild-type (WT) and ΔExon4-deficient fibroblasts were identified by liquid chromatography-tandem mass spectrometry (MS/MS) proteomics analysis. The proteolytic effects of MMP14 were evaluated in vitro via MS identification of eliminated proteins. The biological functions of MMP14-carrying exosomes were investigated via fusion to endothelial cells and flow cytometric assays. Results: Exosomes isolated from WT and ΔExon4-deficient fibroblasts exhibited similar size distributions and morphologies, although WT fibroblasts secreted a greater amount of exosomes. The protein content, however, was higher in ΔExon4-deficient fibroblast-derived exosomes than in WT fibroblast-derived exosomes. Proteomics analysis revealed that WT-derived exosomes included proteins that regulated cell migration, and ΔExon4 fibroblast-derived exosomes contained additional proteins that were cleaved by MMP14. Conclusion: Our findings suggest that MMP14 expression influences the protein composition of exosomes secreted by corneal fibroblasts, and through those biological components, MMP14 in corneal fibroblasts derived-exosomes may regulate corneal angiogenesis.

OSI-027 alleviates oxaliplatin chemoresistance in gastric cancer cells by suppressing P-gp induction

2020 ◽  
Vol 20 ◽  
Author(s):  
En Xu ◽  
Hao Zhu ◽  
Feng Wang ◽  
Ji Miao ◽  
Shangce Du ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Mammalian Target Of Rapamycin ◽  
Cell Counting ◽  
Gastric Cancer Cells ◽  
Ags Cells ◽  
Dual Inhibitor ◽  
Induced Apoptosis

: Gastric cancer is one of the most common malignancies worldwide and the third leading cause of cancer-related death. In the present study, we investigated the potential activity of OSI-027, a potent and selective mammalian target of rapamycin complex 1/2 (mTOR1/2) dual inhibitor, alone or in combination with oxaliplatin against gastric cancer cells in vitro. Cell counting kit-8 assays and EdU staining were performed to examine the proliferation of cancer cells. Cell cycle and apoptosis were detected by flow cytometry. Western blot was used to detect the elements of the mTOR pathway and Pgp in gastric cancer cell lines. OSI-027 inhibited the proliferation of MKN-45 and AGS cells by arresting the cell cycle in the G0/G1 phase. At the molecular level, OSI-027 simultaneously blocked mTORC1 and mTORC2 activation, and resulted in the downregulation of phosphor-Akt, phpspho-p70S6k, phosphor-4EBP1, cyclin D1, and cyclin-dependent kinase4 (CDK4). Additionally, OSI-027 also downregulated P-gp, which enhanced oxaliplatin-induced apoptosis and suppressed multidrug resistance. Moreover, OSI-027 exhibited synergistic cytotoxic effects with oxaliplatin in vitro, while a P-gp siRNA knockdown significantly inhibited the synergistic effect. In summary, our results suggest that dual mTORC1/mTORC2 inhibitors (e.g., OSI-027) should be further investigated as a potential valuable treatment for gastric cancer.

scholarly journals Effect of Shuangdan Mingmu capsule, a Chinese herbal formula, on oxidative stress-induced apoptosis of pericytes through PARP/GAPDH pathway

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Fujiao Nie ◽  
Jiazhao Yan ◽  
Yanjun Ling ◽  
Zhengrong Liu ◽  
Chaojun Fu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Oxidative Damage ◽  
Damage Model ◽  
B Cell Lymphoma ◽  
Cell Counting ◽  
Network Pharmacology ◽  
Diabetic Patients ◽  
Induced Apoptosis

Abstract Background Diabetic retinopathy (DR) has become a worldwide concern because of the rising prevalence rate of diabetes mellitus (DM). Despite much energy has been committed to DR research, it remains a difficulty for diabetic patients all over the world. Since apoptosis of retinal microvascular pericytes (RMPs) is the early characteristic of DR, this study aimed to reveal the mechanism of Shuangdan Mingmu (SDMM) capsule, a Chinese patent medicine, on oxidative stress-induced apoptosis of pericytes implicated with poly (ADP-ribose) polymerase (PARP) / glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pathway. Methods Network pharmacology approach was performed to predict biofunction of components of SDMM capsule dissolved in plasma on DR. Both PARP1 and GAPDH were found involved in the hub network of protein-protein interaction (PPI) of potential targets and were found to take part in many bioprocesses, including responding to the regulation of reactive oxygen species (ROS) metabolic process, apoptotic signaling pathway, and response to oxygen levels through enrichment analysis. Therefore, in vitro research was carried out to validate the prediction. Human RMPs cultured with media containing 0.5 mM hydrogen oxide (H2O2) for 4 h was performed as an oxidative-damage model. Different concentrations of SDMM capsule, PARP1 inhibitor, PARP1 activation, and GAPDH inhibitor were used to intervene the oxidative-damage model with N-Acetyl-L-cysteine (NAC) as a contrast. Flow cytometry was performed to determine the apoptosis rate of cells and the expression of ROS. Cell counting kit 8 (CCK8) was used to determine the activity of pericytes. Moreover, nitric oxide (NO) concentration of cells supernatant and expression of endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), B cell lymphoma 2 (BCL2), vascular endothelial growth factor (VEGF), endothelin 1 (ET1), PARP1, and GAPDH were tested through RT-qPCR, western blot (WB), or immunocytochemistry (ICC). Results Overproduction of ROS, high apoptotic rate, and attenuated activity of pericytes were observed after cells were incubated with media containing 0.5 mM H2O2. Moreover, downregulation of SOD, NO, BCL2, and GAPDH, and upregulation of VEGFA, ET1, and PARP1 were discovered after cells were exposed to 0.5 mM H2O2 in this study, which could be improved by PARP1 inhibitor and SDMM capsule in a dose-dependent way, whereas worsened by PARP1 activation and GAPDH inhibitor. Conclusions SDMM capsule may attenuate oxidative stress-induced apoptosis of pericytes through downregulating PARP expression and upregulating GAPDH expression.

scholarly journals Interleukin 8 Elicits Rapid Physiological Changes in Neutrophils That Are Altered by Inflammatory Conditions

2021 ◽  
pp. 1-17
Author(s):  
Stefan Bernhard ◽  
Stefan Hug ◽  
Alexander Elias Paul Stratmann ◽  
Maike Erber ◽  
Laura Vidoni ◽  
...  
Keyword(s):  
Systemic Inflammation ◽  
Severe Trauma ◽  
Induced Response ◽  
Neutrophil Granulocytes ◽  
Inflammatory Conditions ◽  
Flow Cytometric ◽  
Time Flow ◽  
Flow Cytometric Measurement ◽  
Detailed Kinetics

A sufficient response of neutrophil granulocytes stimulated by interleukin (IL)-8 is vital during systemic inflammation, for example, in sepsis or severe trauma. Moreover, IL-8 is clinically used as biomarker of inflammatory processes. However, the effects of IL-8 on cellular key regulators of neutrophil properties such as the intracellular pH (pH<sub>i</sub>) in dependence of ion transport proteins and during inflammation remain to be elucidated. Therefore, we investigated in detail the fundamental changes in pH<sub>i</sub>, cellular shape, and chemotactic activity elicited by IL-8. Using flow cytometric methods, we determined that the IL-8-induced cellular activity was largely dependent on specific ion channels and transporters, such as the sodium-proton exchanger 1 (NHE1) and non-NHE1-dependent sodium flux. Exposing neutrophils in vitro to a proinflammatory micromilieu with N-formyl-Met-Leu-Phe, LPS, or IL-8 resulted in a diminished response regarding the increase in cellular size and pH. The detailed kinetics of the reduced reactivity of the neutrophil granulocytes could be illustrated in a near-real-time flow cytometric measurement. Last, the LPS-mediated impairment of the IL-8-induced response in neutrophils was confirmed in a translational, animal-free human whole blood model. Overall, we provide novel mechanistic insights for the interaction of IL-8 with neutrophil granulocytes and report in detail about its alteration during systemic inflammation.

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