scholarly journals Investigating FlowSight® imaging flow cytometry as a platform to assess chemically induced micronuclei using human lymphoblastoid cells in vitro

Mutagenesis ◽  
2018 ◽  
Vol 33 (4) ◽  
pp. 283-289 ◽  
Author(s):  
Jatin R Verma ◽  
Danielle S G Harte ◽  
Ume-Kulsoom Shah ◽  
Huw Summers ◽  
Catherine A Thornton ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lymphoblastoid Cells ◽  
Imaging Flow Cytometry ◽  
Chemically Induced

scholarly journals β2M Signals Monocytes Through Non-Canonical TGFβ Receptor Signal Transduction

2021 ◽  
Author(s):  
Zachary T Hilt ◽  
Preeti Maurya ◽  
Laura Tesoro ◽  
Daphne N Pariser ◽  
Sara K Ture ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Flow Cytometry ◽  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Dependent Manner ◽  
Elevated Plasma ◽  
Imaging Flow Cytometry ◽  
Tgfβ Receptor

Rationale: Circulating monocytes can have pro-inflammatory or pro-reparative phenotypes. The endogenous signaling molecules and pathways that regulate monocyte polarization in vivo are poorly understood. We have shown that platelet derived beta-2 microglobulin (β2M) and transforming growth factor beta (TGFβ) have opposing effects on monocytes by inducing inflammatory and reparative phenotypes respectively, but each bind and signal through the same receptor. We now define the signaling pathways involved. Objective: To determine the molecular mechanisms and signal transduction pathways by which β2M and TGFβ regulate monocyte responses both in vitro and in vivo. Methods and Results: Wild-type (WT) and platelet specific β2M knockout (Plt-β2M -/- ) mice were treated intravenously with either β2M or TGFβ to increase plasma concentrations to those in cardiovascular diseases. Elevated plasma β2M increased pro-inflammatory monocytes, while increased plasma TGFβ increased pro-reparative monocytes. TGFβ receptor (TGFβR) inhibition blunted monocyte responses to both β2M and TGFβ in vivo. Using imaging flow cytometry, we found that β2M decreased monocyte SMAD2/3 nuclear localization, while TGFβ promoted SMAD nuclear translocation, but decreased non-canonical/inflammatory (JNK and NFκB nuclear localization). This was confirmed in vitro using both imaging flow cytometry and immunoblots. β2M, but not TGFβ, promoted ubiquitination of SMAD3 and SMAD4, that inhibited their nuclear trafficking. Inhibition of ubiquitin ligase activity blocked non-canonical SMAD-independent monocyte signaling and skewed monocytes towards a pro-reparative monocyte response. Conclusions: Our findings indicate that elevated plasma β2M and TGFβ dichotomously polarize monocytes. Furthermore, these immune molecules share a common receptor, but induce SMAD-dependent canonical signaling (TGFβ) versus non-canonical SMAD-independent signaling (β2M) in a ubiquitin ligase dependent manner. This work has broad implications as β2M is increased in several inflammatory conditions, while TGFβ is increased in fibrotic diseases.

scholarly journals High-throughput multicolor 3D localization in live cells by depth-encoding imaging flow cytometry

2019 ◽  
Author(s):  
Lucien E. Weiss ◽  
Yael Shalev Ezra ◽  
Sarah E. Goldberg ◽  
Boris Ferdman ◽  
Yoav Shechtman
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Imaging System ◽  
Live Cells ◽  
Flow Profile ◽  
Imaging Flow Cytometry ◽  
3D Localization ◽  
Large Populations ◽  
Point Spread Function Engineering

ABSTRACTImaging flow cytometry replaces the canonical point-source detector of flow cytometry with a camera, unveiling subsample details in 2D images while maintaining high-throughput. Here we show that the technique is inherently compatible with 3D localization microscopy by point-spread-function engineering, namely the encoding of emitter depth in the emission pattern captured by a camera. By exploiting the laminar-flow profile in microfluidics, 3D positions can be extracted from cells or other objects of interest by calibrating the depth-dependent response of the imaging system using fluorescent microspheres mixed with the sample buffer. We demonstrate this approach for measuring fluorescently-labeled DNA in vitro and the chromosomal compaction state in large populations of live cells, collecting thousands of samples each minute. Furthermore, our approach is fully compatible with existing commercial apparatus, and can extend the imaging volume of the device, enabling faster flowrates thereby increasing throughput.

scholarly journals Kinetics and Specificity of HEK293T Extracellular Vesicle Uptake using Imaging Flow Cytometry

2020 ◽  
Author(s):  
Brian Jurgielewicz ◽  
Yao Yao ◽  
Steven L. Stice
Keyword(s):  
Flow Cytometry ◽  
Genetic Material ◽  
Cell Types ◽  
Extracellular Vesicle ◽  
Human Neural Stem Cells ◽  
Imaging Flow Cytometry ◽  
Gene Therapy Vectors ◽  
Time Variables

Abstract Background : Extracellular vesicles (EVs) are nanosized vesicles naturally secreted from cells responsible for intercellular communication and delivery of proteins, lipids, and other genetic material. Ultimately, EVs could provide innate therapeutic contents and loaded therapeutic payloads such as small molecules and gene therapy vectors to recipient cells. However, comparative kinetic measures that can be used to quantify and ultimately optimize delivery and uptake of EV payloads are lacking. We investigated both dose and time effects on EV uptake and evaluated the potential specificity of EV uptake to better understand the kinetics and uptake of human embryonic kidney (HEK293T) derived EVs. Results : Utilizing an imaging flow cytometry platform (IFC), HEK293T EV uptake was analyzed. HEK293T EV uptake was dose and time dependent with a minimum threshold dose of 6,000 EVs per cell at 4 hours of co-culture. HEK293T EV uptake was inhibited when co-cultured with recipient cells at 4°C or with pre-fixed recipient cells. By co-culturing HEK293T EVs with cell lines from various germ layers, HEK293T EVs were taken up at higher quantities by HEK293T cells. Lastly, human neural stem cells (hNSCs) internalized significantly more HEK293T EVs relative to mature neurons. Conclusions : Imaging flow cytometry is a quantitative, high throughput, and versatile platform to quantify the kinetics of EV uptake. Utilizing this platform, dose and time variables have been implicated to affect EV uptake measurements making standardization of in vitro and in vivo assays vital for the translation of EVs into the clinic. In this study, we quantified the selectivity of EV uptake between a variety of cell types in vitro and found that EVs were internalized at higher quantities by cells of the same origin. The characterization of HEK293T EV uptake in vitro, notably specificity, dose response, and kinetic assays should be used to help inform and develop EV based therapeutics.

scholarly journals In vitro analysis of antigen induced T cell-monocyte conjugates by imaging flow cytometry

2018 ◽  
Vol 460 ◽  
pp. 93-100
Author(s):  
Meseret Habtamu ◽  
Markos Abebe ◽  
Abraham Aseffa ◽  
Anne Margarita Dyrhol-Riise ◽  
Anne Spurkland ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
Imaging Flow Cytometry ◽  
Vitro Analysis ◽  
In Vitro Analysis

scholarly journals Comparative Study of MSCA-1 and CD146 Isolated Periosteal Cell Subpopulations

2018 ◽  
Vol 51 (3) ◽  
pp. 1193-1206 ◽  
Author(s):  
Felix Umrath ◽  
Carla Thomalla ◽  
Simone Pöschel ◽  
Katja Schenke-Layland ◽  
Siegmar Reinert ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Calcium Phosphate ◽  
Multispectral Imaging ◽  
Osteogenic Potential ◽  
In Vitro Differentiation ◽  
Imaging Flow Cytometry ◽  
Periosteal Cell ◽  
Cell Subpopulations ◽  
Cd146 Expression

Background/Aims: Periosteal tissue is a valuable source of multipotent stem cells for bone tissue engineering. To characterize these cells in detail, we generated an immortalized human cranial periosteal cell line and observed an increased MSCA-1 and CD146 expression, as well as an earlier and stronger mineralization compared to the parental cells. Further, we detected a higher osteogenic potential of MSCA-1high compared to MSCA-1low cranial periosteal cell (CPC) fractions. In the present study, a possible synergism of MSCA-1 and CD146 for periosteal cell mineralization was investigated. Methods: MSCA-1/CD146 positive and negative CPCs were magnetically isolated (MACS) or sorted by flow cytometry (FACS) and subjected to osteogenic differentiation. The expression of osteogenic marker genes in the four subpopulations was analyzed by quantitative real-time PCR. Furthermore, the co-expression of osteogenic markers/antigens was analyzed by multispectral imaging flow cytometry (ImageStream, AMNIS). The mineralization potential was assessed by the quantification of alizarin stainings. Results: While the total cell yield after separation was higher using MACS compared to the FACS approach, the isolation of MSCA-1+/- and CD146+/- subpopulations was more efficient with the FACS separation. The accuracy of the FACS separation of the four distinguished cell subpopulations was confirmed by multispectral imaging flow cytometry. Further, we detected increasing levels of MSCA-1 and CD146 during in vitro differentiation in all subpopulations. However, MSCA-1 expression was significantly higher in the MSCA-1+/CD146+ and MSCA-1+/ CD146- subpopulations, while CD146 expression remained clearly lower in these fractions. Significantly higher gene expression levels of osteogenic markers, ALP and RUNX2, were detected in MSCA-1+ compared to MSCA-1- CPCs at different time points during in vitro differentiation. Staining and quantification of calcium phosphate precipitates revealed a significantly higher mineralization potential of MACS separated MSCA-1+ and CD146- CPCs, compared to their respective counterparts. FACS sorted CPCs displayed earlier mineralization in both MSCA-1+ fractions (d13), while later (d28) only the CD146+/MSCA-1- fraction had a significantly lower calcium phosphate concentration compared to all other fractions. Conclusion: Our results demonstrate, that MSCA-1+ cells isolated from CPCs represent a subpopulation with a higher osteogenic potential. In contrast, we found a lower osteogenic potential in CD146+ CPCs. In conclusion, only MSCA-1, but not CD146, is a suitable marker for the isolation of osteoprogenitors from CPCs.

scholarly journals Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning

2021 ◽  
Author(s):  
John W. Wills ◽  
Jatin R. Verma ◽  
Benjamin J. Rees ◽  
Danielle S. G. Harte ◽  
Qiellor Haxhiraj ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Deep Learning ◽  
Image Classification ◽  
Micronucleus Assay ◽  
Scoring Method ◽  
Safety Testing ◽  
Imaging Flow Cytometry ◽  
Unseen Data ◽  
In Vitro Micronucleus Assay

AbstractThe in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25–5.0 μg/mL) and/or carbendazim (0.8–1.6 μg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the “DeepFlow” neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for ‘mononucleates’, ‘binucleates’, ‘mononucleates with MN’ and ‘binucleates with MN’, respectively. Successful classifications of ‘trinucleates’ (90%) and ‘tetranucleates’ (88%) in addition to ‘other or unscorable’ phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.

scholarly journals Rapid Quantification of NETs In Vitro and in Whole Blood Samples by Imaging Flow Cytometry

2019 ◽  
Vol 95 (5) ◽  
pp. 565-578 ◽  
Author(s):  
Patrick M. Lelliott ◽  
Masatoshi Momota ◽  
Michelle S.J. Lee ◽  
Etsushi Kuroda ◽  
Norifumi Iijima ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Blood Samples ◽  
Imaging Flow Cytometry ◽  
Whole Blood Samples ◽  
Rapid Quantification

scholarly journals Enucleation of primitive erythroid cells generates a transient population of “pyrenocytes” in the mammalian fetus

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2409-2417 ◽  
Author(s):  
Kathleen E. McGrath ◽  
Paul D. Kingsley ◽  
Anne D. Koniski ◽  
Rebecca L. Porter ◽  
Timothy P. Bushnell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Multispectral Imaging ◽  
Fetal Liver ◽  
Fetal Blood ◽  
Immunohistochemical Examination ◽  
Imaging Flow Cytometry ◽  
Transient Population ◽  
Erythroid Maturation ◽  
Definitive Erythropoiesis

Enucleation is the hallmark of erythropoiesis in mammals. Previously, we determined that yolk sac–derived primitive erythroblasts mature in the bloodstream and enucleate between embryonic day (E)14.5 and E16.5 of mouse gestation. While definitive erythroblasts enucleate by nuclear extrusion, generating reticulocytes and small, nucleated cells with a thin rim of cytoplasm (“pyrenocytes”), it is unclear by what mechanism primitive erythroblasts enucleate. Immunohistochemical examination of fetal blood revealed primitive pyrenocytes that were confirmed by multispectral imaging flow cytometry to constitute a distinct, transient cell population. The frequency of primitive erythroblasts was higher in the liver than the bloodstream, suggesting that they enucleate in the liver, a possibility supported by their proximity to liver macrophages and the isolation of erythroblast islands containing primitive erythroblasts. Furthermore, primitive erythroblasts can reconstitute erythroblast islands in vitro by attaching to fetal liver–derived macrophages, an association mediated in part by α4 integrin. Late-stage primitive erythroblasts fail to enucleate in vitro unless cocultured with macrophage cells. Our studies indicate that primitive erythroblasts enucleate by nuclear extrusion to generate erythrocytes and pyrenocytes and suggest this occurs in the fetal liver in association with macrophages. Continued studies comparing primitive and definitive erythropoiesis will lead to an improved understanding of terminal erythroid maturation.

scholarly journals BIOM-25. IDENTIFYING EXTRACELLULAR VESICLES FROM GLIOBLASTOMA OR NON-NEOPLASTIC CELLS VIA IMAGING FLOW CYTOMETRY

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii7-ii7
Author(s):  
Luz Milbeth Cumba Garcia ◽  
Abudumijiti Aibaidula ◽  
Nazanin Yeganeh Kazemi ◽  
Miyeon Jung ◽  
Fabrice Lucien-Matteoni ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Extracellular Vesicles ◽  
Protoporphyrin Ix ◽  
Molecular Profile ◽  
Cell Of Origin ◽  
Double Positive ◽  
Neoplastic Cells ◽  
Monocytic Cell ◽  
Imaging Flow Cytometry

Abstract Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunosuppressive monocytes are heavily infiltrated in these tumors and in patients’ circulation. Treatment-related pseudo-progression confounds outcome assessment by MRI. Thus, there is a need for additional non-invasive methods to assess treatment response. Extracellular vesicles (EVs) contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. We had found significant differences in plasma EVs molecular profile (i.e. miRNA signatures) between GBM patients and healthy donors. Our overall hypothesis is that these differences reflect the EVs cell of origin. Our goal in this project was to develop a fluorescent staining paradigm by flow cytometry to distinguish EVs from different cells in vitro and determine differences in EV miRNA expression profile between GBM and monocytic cell-derived EVs. Gleolan (5-ALA) is an FDA-approved orally available agent for fluorescence-guided resection of GBM tumors. It is metabolized to protoporphyrin IX (PpIX) in GBM cells but not in non-neoplastic cells and has been reported to aid in the detection of GBM-derived EVs by flow cytometry. However, distinguishing between GBM-derived EVs and EVs from other cells of origin has not been described. We co-cultured human GBM cells (dBT114 or dBT116) and CD14+ monocytes for 72 hours in the presence or absence of 5-ALA. EVs were isolated by ultracentrifugation and stained for CD11b (myeloid cell marker). ImageStream Imaging Flow Cytometry was performed showing clear differentiation between PpIX+ EVs from GBM cells and CD11b+ EVs from monocytes. Interestingly, a small number of double-positive EVs (presumably representing monocyte-derived EVs that had taken up PpIX after phagocytizing GBM cells) were also present. Taken together, we were able to optimize a technique to distinguish EVs originating from GBM and monocytes for further characterization by short non-coding RNA sequencing.

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