scholarly journals New Technologies for Use in Toxicology Studies: Monitoring the Effects of Xenobiotics on Immune Function

1990 ◽  
Vol 9 (3) ◽  
pp. 303-317 ◽  
Author(s):  
J. Paul Robinson ◽  
R.W. Pfeifer
Keyword(s):  
Flow Cytometry ◽  
New Technologies ◽  
Single Cells ◽  
Specific Cell ◽  
Kinetic Measurements ◽  
New Developments ◽  
Cell Functions ◽  
Alternative Approach ◽  
In Vitro Systems

New developments in flow cytometry are now being applied in toxicology studies. There are several reasons for using this technology. First, techniques are well characterized to measure functional parameters of single cells. Such measurements can be directly related to perturbations by xenobiotics, cell-mediated immune responses, or trauma. Second, there is a clear indication for movement toward in vitro systems as highly objective assessments of toxicologic interactions. By measuring specific cell functions at the single cell level, it is possible to define a range of normal responses. More importantly, a multiparametric analysis can be performed with flow cytometry and parameters can be directly related to one another. Furthermore, kinetic measurements can be made, providing vital clues to the mechanisms of actions of drugs or chemicals on functions of specific cell populations. Major advantages of this approach are that studies can be performed on very small volumes of blood, body fluid, or cell culture lines and it is not necessary to isolate pure populations of cells to perform these assays. We believe that this alternative approach in toxicology will provide valuable information unobtainable by traditional means.

scholarly journals Functional angiogenesis requires microenvironmental cues balancing endothelial cell migration and proliferation

2019 ◽  
Author(s):  
William Y. Wang ◽  
Daphne Lin ◽  
Evan H. Jarman ◽  
William J. Polacheck ◽  
Brendon M. Baker
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cell ◽  
Single Cells ◽  
Endothelial Cell Migration ◽  
Migration Speed ◽  
Parent Vessel ◽  
Extracellular Milieu ◽  
Cell Functions

ABSTRACTAngiogenesis is a complex morphogenetic process that involves intimate interactions between multicellular endothelial structures and their extracellular milieu. In vitro models of angiogenesis can aid in reducing the complexity of the in vivo microenvironment and provide mechanistic insight into how soluble and physical extracellular matrix cues regulate this process. To investigate how microenvironmental cues regulate angiogenesis and the function of resulting microvasculature, we multiplexed an established angiogenesis-on-a-chip platform that affords higher throughput investigation of 3D endothelial cell sprouting emanating from a parent vessel through defined biochemical gradients and extracellular matrix. We found that two fundamental endothelial cell functions, migration and proliferation, dictate endothelial cell invasion as single cells vs. multicellular sprouts. Microenvironmental cues that elicit excessive migration speed incommensurate with proliferation resulted in microvasculature with poor barrier function and an inability to transport fluid across the microvascular bed. Restoring the balance between migration speed and proliferation rate rescued multicellular sprout invasion, providing a new framework for the design of pro-angiogenic biomaterials that guide functional microvasculature formation for regenerative therapies.

scholarly journals Applications of Nanosheets in Frontier Cellular Research

Nanomaterials ◽  
2018 ◽  
Vol 8 (7) ◽  
pp. 519 ◽  
Author(s):  
Wenjing Huang ◽  
Yuta Sunami ◽  
Hiroshi Kimura ◽  
Sheng Zhang
Keyword(s):  
Graphene Oxide ◽  
Lactic Acid ◽  
Imaging Techniques ◽  
Cell Protein ◽  
Specific Cell ◽  
Protein Sensing ◽  
Cell Functions ◽  
The Past ◽  
Application Prospects

Several types of nanosheets, such as graphene oxide (GO) nanosheet, molybdenum disulfide (MoS2) and poly(l-lactic acid) (PLLA) nanosheets, have been developed and applied in vitro in cellular research over the past decade. Scientists have used nanosheet properties, such as ease of modification and flexibility, to develop new cell/protein sensing/imaging techniques and achieve regulation of specific cell functions. This review is divided into three main parts based on the application being examined: nanosheets as a substrate, nanosheets as a sensitive surface, and nanosheets in regenerative medicine. Furthermore, the applications of nanosheets are discussed, with two subsections in each section, based on their effects on cells and molecules. Finally, the application prospects of nanosheets in cellular research are summarized.

scholarly journals Comparison of cellular Na+ assays by flow fluorometry with ANG-2 and flame emission: Pitfalls in using ionophores for calibrating fluorescent probes

2020 ◽  
Author(s):  
Valentina E. Yurinskaya ◽  
Nikolay D. Aksenov ◽  
Alexey V. Moshkov ◽  
Tatyana S. Goryachaya ◽  
Alexey A. Vereninov
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Probes ◽  
Single Cells ◽  
Intracellular Sodium ◽  
Cell Populations ◽  
Emission Analysis ◽  
Cell Functions ◽  
Flame Emission ◽  
Monovalent Ions ◽  
In Cells

AbstractMonovalent ions, sodium in particular, are involved in fundamental cell functions, such as water balance and electric processes, intra- and intercellular signaling, cell movement, pH regulation and metabolite transport into and out of cells. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells in heterogeneous cell populations and tissues. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ by changing the concentration of extracellular sodium in the presence of ionophores, making the membrane permeable to sodium and equilibrating its intra- and extracellular concentrations. The reliability of this calibration method has not been well studied. Here, we compare the determinations of the intracellular sodium concentration by flame emission photometry and flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG). The intracellular Na+ concentration was altered using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. The use of U937 cells cultured in suspension allowed the fluorometry of single cells by flow cytometry and flame emission analysis of samples checked for uniform cell populations. It is revealed that the ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. Sodium sensitive fluorescent dyes are widely used at present, and the problem is practically significant.

New views inside cells with a digital imaging microscope

1991 ◽  
Vol 49 ◽  
pp. 240-241
Author(s):  
F. S. Fay ◽  
Kevin Fogarty ◽  
Richard Tuft ◽  
Walter Carrington
Keyword(s):  
Digital Imaging ◽  
Cell Biology ◽  
Cell Function ◽  
Single Cells ◽  
Low Noise ◽  
Molecular Composition ◽  
Specific Cell ◽  
Wide Field ◽  
High Quantum Efficiency ◽  
Cell Functions

Many current questions in cell biology revolve around questions regarding how changes in cell function are caused by changes in their molecular composition. Given that cells are highly organized structures often carrying out diverse functions in different compartments, it follows that changes in specific cell functions must involve highly localized changes in molecular composition.We have been involved in the development of the digital imaging microscope as a tool to investigate the distribution of molecules inside single living cells. The system measures fluorescence of probes that are highly fluorescent and specific for a molecule or ion of interest and utilizes a wide-field rather than a confocal microscope to produce an image of fluorescence in a single cell. The image is captured by a high quantum efficiency, low noise cooled CCD, thereby providing ultrahigh efficiency in the acquisition of fluorescent images. By utilizing very powerful light sources, the system is capable of generating an image with good signal-to-noise ratio in a millisecond or less, thereby allowing one to follow extremely rapid changes in molecular or ion distribution in single cells.

scholarly journals An alternative approach to produce versatile retinal organoids with accelerated ganglion cell development

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Philip E. Wagstaff ◽  
Anneloor L. M. A. ten Asbroek ◽  
Jacoline B. ten Brink ◽  
Nomdo M. Jansonius ◽  
Arthur A. B. Bergen
Keyword(s):  
Ganglion Cells ◽  
Cell Types ◽  
Retinal Cell ◽  
Specific Cell ◽  
Retinal Ganglion ◽  
In Vivo Models ◽  
Alternative Approach

AbstractGenetically complex ocular neuropathies, such as glaucoma, are a major cause of visual impairment worldwide. There is a growing need to generate suitable human representative in vitro and in vivo models, as there is no effective treatment available once damage has occured. Retinal organoids are increasingly being used for experimental gene therapy, stem cell replacement therapy and small molecule therapy. There are multiple protocols for the development of retinal organoids available, however, one potential drawback of the current methods is that the organoids can take between 6 weeks and 12 months on average to develop and mature, depending on the specific cell type wanted. Here, we describe and characterise a protocol focused on the generation of retinal ganglion cells within an accelerated four week timeframe without any external small molecules or growth factors. Subsequent long term cultures yield fully differentiated organoids displaying all major retinal cell types. RPE, Horizontal, Amacrine and Photoreceptors cells were generated using external factors to maintain lamination.

scholarly journals Identification and further characterization of the specific cell binding fragment from sponge aggregation factor.

1986 ◽  
Vol 102 (4) ◽  
pp. 1344-1349 ◽  
Author(s):  
M Gramzow ◽  
M Bachmann ◽  
G Uhlenbruck ◽  
A Dorn ◽  
W E Müller
Keyword(s):  
Single Cells ◽  
Sodium Dodecyl ◽  
Cell System ◽  
Specific Cell ◽  
Cell Binding ◽  
Aggregation Factor ◽  
Indirect Immunofluorescence Staining ◽  
Number Of Binding Sites ◽  
Ca Ions

Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia.

Surfaces Designed to Control the Projected Area and Shape of Individual Cells

1999 ◽  
Vol 121 (1) ◽  
pp. 40-48 ◽  
Author(s):  
C. H. Thomas ◽  
J.-B. Lhoest ◽  
D. G. Castner ◽  
C. D. McFarland ◽  
K. E. Healy
Keyword(s):  
Mammalian Cells ◽  
Cell Function ◽  
Single Cells ◽  
Polymer Network ◽  
Interpenetrating Polymer Network ◽  
Specific Cell ◽  
Cell Binding ◽  
Projected Area ◽  
Cell Functions ◽  
Spatially Resolved

Materials with spatially resolved surface chemistry were designed to isolate individual mammalian cells to determine the influence of projected area on specific cell functions (e.g., proliferation, cytoskeletal organization). Surfaces were fabricated using a photolithographic process resulting in islands of cell binding N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS) separated by a nonadhesive interpenetrating polymer network [poly (acrylamide-co-ethylene glycol); P(AAm-co-EG)]. The surfaces contained over 3800 adhesive islands/cm2, allowing for isolation of single cells with projected areas ranging from 100 μm2 to 10,000 μm2. These surfaces provide a useful tool for researching how cell morphology and mechanical forces affect cell function.

scholarly journals Immune cells lacking Y chromosome show dysregulation of autosomal gene expression

2021 ◽  
Author(s):  
Jan P. Dumanski ◽  
Jonatan Halvardson ◽  
Hanna Davies ◽  
Edyta Rychlicka-Buniowska ◽  
Jonas Mattisson ◽  
...  
Keyword(s):  
Single Cells ◽  
Cell Types ◽  
Autosomal Gene ◽  
Specific Cell ◽  
Immune Functions ◽  
Chromosome Y ◽  
Late Genes ◽  
Increased Risk

AbstractEpidemiological investigations show that mosaic loss of chromosome Y (LOY) in leukocytes is associated with earlier mortality and morbidity from many diseases in men. LOY is the most common acquired mutation and is associated with aberrant clonal expansion of cells, yet it remains unclear whether this mosaicism exerts a direct physiological effect. We studied DNA and RNA from leukocytes in sorted- and single-cells in vivo and in vitro. DNA analyses of sorted cells showed that men diagnosed with Alzheimer’s disease was primarily affected with LOY in NK cells whereas prostate cancer patients more frequently displayed LOY in CD4 + T cells and granulocytes. Moreover, bulk and single-cell RNA sequencing in leukocytes allowed scoring of LOY from mRNA data and confirmed considerable variation in the rate of LOY across individuals and cell types. LOY-associated transcriptional effect (LATE) was observed in ~ 500 autosomal genes showing dysregulation in leukocytes with LOY. The fraction of LATE genes within specific cell types was substantially larger than the fraction of LATE genes shared between different subsets of leukocytes, suggesting that LOY might have pleiotropic effects. LATE genes are involved in immune functions but also encode proteins with roles in other diverse biological processes. Our findings highlight a surprisingly broad role for chromosome Y, challenging the view of it as a “genetic wasteland”, and support the hypothesis that altered immune function in leukocytes could be a mechanism linking LOY to increased risk for disease.

scholarly journals Immunogenic effects of recombinant interferon-beta therapy disrupt the JAK/STAT pathway in primary immune cells from patients with multiple sclerosis

2012 ◽  
Vol 18 (8) ◽  
pp. 1116-1124 ◽  
Author(s):  
S Gavasso ◽  
BT Gjertsen ◽  
E Anderssen ◽  
KM Myhr ◽  
C Vedeler
Keyword(s):  
Multiple Sclerosis ◽  
Flow Cytometry ◽  
Neutralizing Antibodies ◽  
Mononuclear Cells ◽  
Specific Cell ◽  
Peripheral Blood Mononuclear ◽  
Specific Flow ◽  
Recombinant Interferon ◽  
The Impact

Background: Immunogenicity of recombinant interferon-β (IFN-β) is a known complication in the therapy of relapsing–remitting multiple sclerosis (RRMS). Neutralizing antibodies (NAbs) that can interfere with efficacy are quantified using in vitro bioassays; however, these assays do not reveal the immunogenic state of the patient and are not predictive of treatment outcome. Objective: Assessment of the impact of NAbs on IFN-β responsive cells and signalling pathways in peripheral blood mononuclear cells (PBMCs) with phospho-specific flow cytometry. Method: PBMCs from 10 IFN-β-treated patients with RRMS, two untreated patients, and two healthy controls were re-stimulated in autologous sera and media with a serial dilution of IFN-β (0–8000 U/ml) and levels of phosphorylation of STAT1/3/4/5/6 transcription factors were quantified in PBMC subtypes (NAb titres 0 to > 6000 neutralizing units). Data was subjected to principal component analysis, Hotelling’s T2, and partial least squares analysis. Results: Three significantly distinct clusters of individuals were revealed in autologous sera: therapy-naïve and healthy, treated NAb-negative, and treated NAb-positive. Compared with controls STATs signalling patterns were modulated in treated NAb-negative patients and inhibited in all treated NAb-positive patients independently of NAb titres. In media no clustering of patients could be found. The predictability of NAb titres based on the phospho-flow data was 74%. Conclusion: Phospho-specific flow cytometry can delineate subset-specific cell responses that can act as surrogates for NAb exposure in blood. Immunogenic effects alter the response in primary cells even at low NAb levels. Cell line-based immunogenicity testing is not readily transferable to the immunogenic response in patients.

Functional Studies on the C-Type Lectin Receptor CD302 Present on Dendritic Cells and Macrophages

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2198-2198
Author(s):  
Derek NJ Hart ◽  
Pablo Silveira ◽  
Tsun Ho Lo ◽  
Nirupama Verma ◽  
Ai Vu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Dendritic Cells ◽  
Immune Cell ◽  
Blood Monocytes ◽  
Lectin Receptors ◽  
Functional Studies ◽  
Cell Functions ◽  
Equity Ownership

Abstract Introduction: C-type lectin receptors (CLR) play an important role in the immune system by recognising molecular patterns expressed by exogenous and endogenous threats. They have been shown to capture and internalise antigens and to mediate other important immune cell functions. DEC205 and CLEC9A are being actively investigated as targets for clinical therapeutic cancer vaccines. We discovered CD302 as a new CLR expressed on human dendritic cells (DC), monocytes and macrophages (J Immunol 2007;179:6052). Our initial studies suggested the molecule could play a role in cell adhesion or migration due to its co-localisation with migratory structures on macrophages. Our study set out to investigate the potential immunological function of CD302 using mouse models and to define its wider tissue expression in man. Methods: We generated CD302 knockout (KO) mice lacking exon 1 of its gene, abrogating transcription, for functional studies. We characterised the transcriptional expression of CD302 in mouse immune cells using real-time PCR. We developed monoclonal mAb to mCD302. Human studies utilized the anti-CD302 mAbs, MMRI-20 & 21 in flow cytometry and confocal microscopy studies of human immune cell populations. Results: CD302 was primarily expressed in mouse liver, lungs, lymph nodes (LN) and spleen. In spleen, macrophages, granulocytes and dendritic cells (DC) expressed CD302. Analysis of LN DC subsets revealed 2.5-fold higher CD302 mRNA expression in migratory compared to resident DC populations. Enumeration of various immune populations in lymphoid organs by flow cytometry uncovered a modest deficiency in migratory DC number and proportion within LN of CD302 KO mice compared to wild-type (WT) mice. In vitro studies showed CD302 KO and WT DC had an equivalent capacity to be activated by various stimuli, prime T cells and migrate towards the lymphoid-homing chemokines CCL19/CCL21. CD302 KO migratory DC exhibited a reduced in vivo migratory capacity to LN after FITC skin-painting. However, CD302 KO macrophages migrated similarly to WT macrophages in vivo in response to thioglycollate. In man, CD302 was present in high density in liver and peripheral blood monocytes and myeloid but not plasmacytoid DC. Current studies are aimed at clarifying its distribution on tissue DC and macrophage subsets. Anti-CD302 coated microbeads were taken up by human monocyte derived macrophages and anti-CD302 mAb was also internalized by DC. Confocal studies showed that CD302 co-localized with F-actin structures at the near basal surface such as filopodia and lamellipodia and podosomes of human macrophages and EGFP tagged CD302 expressed in COS-1 cells associated with F-actin. Conclusion: Our data suggests that CD302 may play a specialist role in DC and macrophage membrane functions. This appears to relate to its ability to associate with F-actin and may contribute to the membrane interactions required for DC to migrate towards the draining LN. Disclosures Hart: DendroCyte BioTech Pty Ltd: Equity Ownership. Clark:DendroCyte BioTech Pty Ltd: Equity Ownership.

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