scholarly journals Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells

2020 ◽  
Vol 11 (3) ◽  
pp. 702-715 ◽  
Author(s):  
Michael Gorry ◽  
Toshie Yoneyama ◽  
Lazar Vujanovic ◽  
Marcia L. Moss ◽  
Michelle A. Garlin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Enzyme Activity ◽  
Cell Membrane ◽  
Measuring Cell ◽  
Membrane Enzyme

Surface chemistry modification of silica nanoparticles alters the activation of monocytes

2021 ◽  
Author(s):  
Romina Mitarotonda ◽  
Martín Saraceno ◽  
Marcos Todone ◽  
Exequiel Giorgi ◽  
Emilio L Malchiodi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune System ◽  
Cell Membrane ◽  
Cell Activation ◽  
Membrane Damage ◽  
Living Cells ◽  
Cell Membrane Damage ◽  
Study Materials ◽  
Therapeutic Molecules ◽  
Pro Inflammatory Cytokines

Aim: Nanoparticles (NPs) interaction with immune system is a growing topic of study. Materials & methods: Bare and amine grafted silica NPs effects on monocytes/macrophages cells were analyzed by flow cytometry, MTT test and LIVE/DEAD® viability/cytotoxicity assay. Results: Bare silica NPs inhibited proliferation and induced monocyte/macrophages activation (increasing CD40/CD80 expression besides pro-inflammatory cytokines and nitrite secretion). Furthermore, silica NPs increased cell membrane damage and reduced the number of living cells. In contrast, amine grafted silica NPs did not alter these parameters. Conclusion: Cell activation properties of bare silica NPs could be hindered after grafting with amine moieties. This strategy is useful to tune the immune system stimulation by NPs or to design NPs suitable to transport therapeutic molecules.

scholarly journals Susceptibility of melanized and nonmelanized Cryptococcus neoformans to the melanin-binding compounds trifluoperazine and chloroquine.

1996 ◽  
Vol 40 (3) ◽  
pp. 541-545 ◽  
Author(s):  
Y Wang ◽  
A Casadevall
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Antipsychotic Agent ◽  
Measuring Cell ◽  
High Affinity ◽  
Propidium Iodide Staining ◽  
Opportunistic Fungal Pathogen ◽  
Melanin Binding

Cryptococcus neoformans is an opportunistic fungal pathogen which becomes heavily melanized in the presence of phenolic substrates such as L-dopa. Various drugs are known to bind to melanin with high affinity, including the antipsychotic agent trifluoperazine and the antimalarial agent chloroquine. We hypothesized that drugs which bind melanin may have different toxicities for melanized and nonmelanized C. neoformans cells. The effects of trifluoperazine and chloroquine or C. neoformans were determined by measuring cell viability after exposure to these drugs. Cell viability was measured by CFU determination and flow cytometry with propidium iodide staining. Melanized cells were more susceptible than nonmelanized cells to the fungicidal effects of trifluoperazine. Chloroquine had no fungicidal effect on either melanized or nonmelanized C. neoformans under the conditions studied. Flow cytometry of trifluoperazine-treated C. neoformans cells stained with the mitochondrial stain dihydrorhodamine 123 revealed fluorescence changes consistent with mitochondrial damage. Our results indicate that melanized and nonmelanized C. neoformans cells can differ in susceptibility to certain drugs and suggest that strategies which target melanin may be productive for antifungal-drug discovery.

scholarly journals Heterogeneity in the Rate of Benzo[a]pyrene Metabolism in Single Cells: Quantitation Using Flow Cytometry

1982 ◽  
Vol 2 (6) ◽  
pp. 625-632 ◽  
Author(s):  
Arthur G. Miller ◽  
James P. Whitlock
Keyword(s):  
Flow Cytometry ◽  
Enzyme Activity ◽  
Cell Line ◽  
Single Cells ◽  
Hepatoma Cells ◽  
Microsomal Enzyme ◽  
Clonal Population ◽  
Enzyme Inducer ◽  
Mouse Hepatoma ◽  
Low Activity

We describe a method for quantitating heterogeneity in the rate of benzo[a]pyrene metabolism in single cells by using flow cytometry. We have used the technique to study the response of Hepa-1c1c7 mouse hepatoma cells to the microsomal enzyme inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin. Cells responded in a relatively homogeneous fashion at different times of induction with a maximally inducing concentration of the inducer. However, the induction response could be heterogeneous at a submaximal inducer concentration. We found even higher heterogeneity of enzyme activity among low-activity variants derived from the Hepa-1c1c7 cell line. When cells of either high or low activity were isolated from such a clonal population, propagated, and reanalyzed, they displayed average enzyme activity and heterogeneity identical to the parental cells; therefore, the heterogeneity represents transient, nonheritable differences between cells within the population.

Membrane Remodeling By Pathogenic Antibodies Underlies Monocyte Activation in Heparin-Induced Thrombocytopenia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2244-2244
Author(s):  
Izabella Andrianova ◽  
Vincent M Hayes ◽  
Daria Madeeva ◽  
Rustem I. Litvinov ◽  
Douglas B. Cines ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Cell Membrane ◽  
Cell Activation ◽  
Monocyte Activation ◽  
Heparin Induced Thrombocytopenia ◽  
Scanning Electron

Abstract Heparin induced thrombocytopenia (HIT) is an iatrogenic antibody-mediated disorder with a paradoxically high propensity for thrombosis. We have shown previously that human HIT IgGs and the HIT-like monoclonal antibody (MAb) KKO bind to platelet factor 4 (PF4) complexed with glycosaminoglycans (GAGs) on the surface of platelets and monocytes, initiating cell activation in vitro, thrombocytopenia in a transgenic mouse model, and thrombus formation in a laser microvascular injury model in vivo even in the absence of exogenous heparin. Monocytes bind PF4 and HIT Ab more readily than platelets because they express higher affinity GAGs, heparan sulfate and dermatan sulfate, in addition to chondroitin sulfate found on both cell types. To study changes in the structure of the monocytes that accompany HIT, we used scanning electron microscopy, confocal microscopy and flow cytometry to characterize the morphology and function of isolated human monocytes and mouse transgenic Fcg receptor IIA positive (FcγRIIA+) or wt (FcγRIIA-) monocytes in the absence or presence of platelets. We show by scanning electron microscopy that upon binding of pathogenic HIT Abs to PF4/GAG complexes on FcgRIIA expressing monocytes, they initiate profound remodeling of the cell membrane. Addition of 100 μg/ml recombinant human PF4 in the absence of HIT Abs initiates the activation process with the appearance of 177 ± 53 nm "knobs" on the surface of 70% of monocytes. Subsequent addition of the HIT-like monoclonal antibody KKO at 50 μg/ml dramatically alters the cellular surface with the appearance of large 701 ± 208 nm membrane "blebs" that were not seen on FcγRIIA-mouse monocytes. These large, membrane-associated structures likely engage FcγRIIA, clustering them in proximity to cell-bound immune complexes, which promotes cell activation that leads to thrombosis. These blebs increase in size over time and are then shed from the cells as monocyte-derived microparticles, which self-aggregate. As a result of shedding of these blebs, the monocytes lose much of their typical ruffled surface (only 67% of monocytes maintain ruffles in the presence of PF4 plus KKO, compared to 97% of control monocytes) and appear smoother, sometimes with pores indicating degranulation. In the presence of platelets, monocytes exposed to PF4 and KKO formed heterocellular aggregates in addition to these subcellular changes. In contrast to KKO, addition of the non-pathogenic MAb RTO not only did not induce blebbing, but largely inhibited PF4-induced changes in the monocyte surface. This suggests that RTO might prevent monocyte activation by interfering with PF4 tetramerization. Structural analysis of the shed microparticles by microscopy revealed that they had an average diameter of 356 ± 307 nm, with many larger particles and aggregates. Flow cytometry confirmed that the shed particles contain cell membrane lipids and receptors. Confocal microscopy showed uniform binding of labeled PF4 to the monocyte cell membrane followed by rapid clustering into large complexes after the addition of KKO, but not RTO. These studies affirm the centrality of cell surface PF4/GAG complexes in the pathogenesis of HIT and provide quantitative morphometric characteristics of the changes in the monocyte membrane structure. We propose that PF4 released from activated platelets binds to the surface of GAG-expressing monocytes in vivo, forming clusters of PF4/GAG complexes that likely promote antibody binding and cause monocyte activation through FcγRIIA along with large-scale remodeling of the cell membrane and shedding of procoagulant microparticles. Disclosures No relevant conflicts of interest to declare.

Electrical Resistance and Volume Flow in Glass Microelectrodes

1971 ◽  
Vol 49 (5) ◽  
pp. 436-447 ◽  
Author(s):  
D. R. Firth ◽  
L. J. DeFelice
Keyword(s):  
Cell Membrane ◽  
Electrical Resistance ◽  
Surface Condition ◽  
External Solution ◽  
Membrane Resistance ◽  
Volume Flow ◽  
Glass Wall ◽  
Measuring Cell ◽  
Cell Membrane Resistance ◽  
Flow Effects

The electrical resistance and the flow of solution in glass microelectrodes has been studied as function of the concentration of internal and external solution (KCl, 3–10−5 M), and as a function of pressure between the inside and outside solutions. The study demonstrates the usefulness of using microelectrodes for measuring cell membrane resistance and also the dangers in using them as microejectors because of anomalous flow effects. Electrokinetic effects and tip potentials are briefly described; they offer a means of investigating the surface condition of the glass wall inside the tip of the electrode and its variation with the outside solution.

Metal-induced alteration of the cell membrane/cytoplasm complex studied by flow cytometry and detergent lysis

Toxicology ◽  
1988 ◽  
Vol 53 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Robert M. Zucker ◽  
Kenneth H. Elstein ◽  
Robert E. Easterling ◽  
Edward J. Massaro
Keyword(s):  
Flow Cytometry ◽  
Cell Membrane
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