pinecone/core-2020-05-10-deduplication huggingface dataset:
A novel flow cytometric protocol for assessment of yeast cell adhesion

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner
Standard fluorescent imaging of live cells is highly genotoxic

Fluorescence microscopy is commonly used for imaging live mammalian cells. Here, we describe studies aimed at revealing the potential genotoxic effects of standard fluorescence microscopy. To assess DNA damage, a high throughput platform for single cell gel electrophoresis is used (e.g., the CometChip). Light emitted by three standard filters was studied: (a) violet light [340–380 nm], used to excite DAPI and other blue fluorophores, (b) blue light [460–500 nm] commonly used to image green fluorescent protein (GFP) and Calcein AM, and (c) green light [528–553 nm], useful for imaging red fluorophores. Results show that exposure of samples to light during imaging is indeed genotoxic even when the selected wavelengths are outside the range known to induce significant damage levels. Shorter excitation wavelengths and longer irradiation times lead to higher levels of DNA damage. We have also measured DNA damage in cells expressing enhanced GFP or stained with Calcein AM, a widely used green fluorophore. Data show that Calcein AM leads to a synergistic increase in the levels of DNA damage and that even cells that are not being directly imaged sustain significant DNA damage from exposure to indirect light. The nature of light-induced DNA damage during imaging was assessed using the Fpg glycosylase, an enzyme that enables quantification of oxidative DNA damage. Oxidative damage was evident in cells exposed to violet light. Furthermore, the Fpg glycosylase revealed the presence of oxidative DNA damage in blue-light exposed cells for which DNA damage was not detected using standard analysis conditions. Taken together, the results of these studies call attention to the potential confounding effects of DNA damage induced by standard imaging conditions, and identify wavelength, exposure time, and fluorophore as parameters that can be modulated to reduce light-induced DNA damage.National Institutes of Health (U.S.) (Grant 5-UO1-ES016045)National Institutes of Health (U.S.) (grant P30-ES002109)National Institutes of Health (U.S.) (Grant 1-R21-ES019498)National Institutes of Health (U.S.) (Grant R43-ES021116-01)National Institute of Environmental Health Sciences (NIEHS Training Grant in Environmental Toxicology number T32-ES007020
High throughput FRET analysis of protein-protein interactions by slide-based imaging laser scanning cytometry.

Laser scanning cytometry (LSC) is a slide-based technique combining advantages of flow and image cytometry: automated, high-throughput detection of optical signals with subcellular resolution. Fluorescence resonance energy transfer (FRET) is a spectroscopic method often used for studying molecular interactions and molecular distances. FRET has been measured by various microscopic and flow cytometric techniques. We have developed a protocol for a commercial LSC instrument to measure FRET on a cell-by-cell or pixel-by-pixel basis on large cell populations, which adds a new modality to the use of LSC. As a reference sample for FRET, we used a fusion protein of a single donor and acceptor (ECFP-EYFP connected by a seven-amino acid linker) expressed in HeLa cells. The FRET efficiency of this sample was determined via acceptor photobleaching and used as a reference value for ratiometric FRET measurements. Using this standard allowed the precise determination of an important parameter (the alpha factor, characterizing the relative signal strengths from a single donor and acceptor molecule), which is indispensable for quantitative FRET calculations in real samples expressing donor and acceptor molecules at variable ratios. We worked out a protocol for the identification of adherent, healthy, double-positive cells based on light-loss and fluorescence parameters, and applied ratiometric FRET equations to calculate FRET efficiencies in a semi-automated fashion. To test our protocol, we measured the FRET efficiency between Fos-ECFP and Jun-EYFP transcription factors by LSC, as well as by confocal microscopy and flow cytometry, all yielding nearly identical results. Our procedure allows for accurate FRET measurements and can be applied to the fast screening of protein interactions. A pipeline exemplifying the gating and FRET analysis procedure using the CellProfiler software has been made accessible at our web site. (c) 2013 International Society for Advancement of Cytometry
A novel method for monitoring tumor proliferation in vivo using fluorescent dye DiD

Monitoring single cell proliferation in vivo is difficult, but optimizing this technique is essential in order to expand our knowledge of the regulation of tumor proliferation. In this study, we used a lipophilic fluorescent dye, DiD, that rapidly and stably integrates into the phospholipid cell membrane. We cultured DiD‐stained prostate cancer cell lines for 10 days and isolated cells by flow cytometry based on expression levels of DiD. We found that a decrease in DiD intensity was correlated to the reduction of EdU, where the DiD‐high population proliferated more slowly than the DiD‐low population and the DiD‐low population exhibited a higher mitotic index. We also found that DiD was detected after 3 weeks of implantation in an in vivo setting. Importantly, DiD dye did not have any effect on normal cell growth, whereas a gold standard fluorescent dye for measuring cell proliferation, CFSE, slowed cell proliferation. Although further study is indicated, DiD can be useful for identifying the molecular mechanisms underlying tumor proliferation in vivo. © 2014 International Society for Advancement of Cytometr
A color and shape based algorithm for segmentation of white blood cells in peripheral blood and bone marrow images

Cataloged from PDF version of article.Computer-based imaging systems are becoming important tools for quantitative assessment
of peripheral blood and bone marrow samples to help experts diagnose blood disorders
such as acute leukemia. These systems generally initiate a segmentation stage
where white blood cells are separated from the background and other nonsalient objects.
As the success of such imaging systems mainly depends on the accuracy of this stage,
studies attach great importance for developing accurate segmentation algorithms.
Although previous studies give promising results for segmentation of sparsely distributed
normal white blood cells, only a few of them focus on segmenting touching and overlapping
cell clusters, which is usually the case when leukemic cells are present. In this article,
we present a new algorithm for segmentation of both normal and leukemic cells in
peripheral blood and bone marrow images. In this algorithm, we propose to model color
and shape characteristics of white blood cells by defining two transformations and introduce
an efficient use of these transformations in a marker-controlled watershed algorithm.
Particularly, these domain specific characteristics are used to identify markers and
define the marking function of the watershed algorithm as well as to eliminate false white
blood cells in a postprocessing step. Working on 650 white blood cells in peripheral
blood and bone marrow images, our experiments reveal that the proposed algorithm
improves the segmentation performance compared with its counterparts, leading to high accuracies for both sparsely distributed normal white blood cells and dense leukemic cell clusters. (C) 2014 International Society for Advancement of Cytometr
3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton

The ELF or fluorescence-labeled enzyme activity (FLEA) technique is a culture-independent single-cell tool for assessing plankton enzyme activity in close-to-in situ conditions. We demonstrate that single-cell FLEA quantifications based on two-dimensional (2D) image analysis were biased by up to one order of magnitude relative to deconvolved 3D. This was basically attributed to out-of-focus light, and partially to object size. Nevertheless, if sufficient cells were measured (25-40 cells), biases in individual 2D cell measurements were partially compensated, providing useful and comparable results to deconvolved 3D. We also discuss how much caution should be used when comparing the single-cell enzyme activities of different sized bacterio- and/or phytoplankton populations measured on 2D images. Finally, a novel method based on deconvolved 3D images (wide field restoration microscopy; WFR) was devised to improve the discrimination of similar single-cell enzyme activities, the comparison of enzyme activities between different size cells, the measurement of low fluorescence intensities, the quantification of less numerous species, and the combination of the FLEA technique with other single-cell methods. These improvements in cell enzyme activity measurements will provide a more precise picture of individual species' behavior in nature, which is essential to understand their functional role and evolutionary history
Three-dimensional image cytometer based on widefield structured light microscopy and high-speed remote depth scanning

A high throughput 3D image cytometer have been developed that improves imaging speed by an order of magnitude over current technologies. This imaging speed improvement was realized by combining several key components. First, a depth-resolved image can be rapidly generated using a structured light reconstruction algorithm that requires only two wide field images, one with uniform illumination and the other with structured illumination. Second, depth scanning is implemented using the high speed remote depth scanning. Finally, the large field of view, high NA objective lens and the high pixelation, high frame rate sCMOS camera enable high resolution, high sensitivity imaging of a large cell population. This system can image at 800 cell/sec in 3D at submicron resolution corresponding to imaging 1 million cells in 20 min. The statistical accuracy of this instrument is verified by quantitatively measuring rare cell populations with ratio ranging from 1:1 to 1:10[superscript 5].National Institutes of Health (U.S.) (Grant 9P41EB015871-26A1)National Institutes of Health (U.S.) (Grant 5R01EY017656-02)National Institutes of Health (U.S.) (Grant 5R01 NS051320)National Institutes of Health (U.S.) (Grant 4R44EB012415-02)National Science Foundation (U.S.) (Grant CBET-0939511)Singapore-MIT Alliance for Research and TechnologyMIT Skoltech InitiativeHamamatsu CorporationDavid H. Koch Institute for Integrative Cancer Research at MIT (Bridge Project Initiative
Identification and visualization of multidimensional antigen-specific T-cell populations in polychromatic cytometry data.

An important aspect of immune monitoring for vaccine development, clinical trials, and research is the detection, measurement, and comparison of antigen-specific T-cells from subject samples under different conditions. Antigen-specific T-cells compose a very small fraction of total T-cells. Developments in cytometry technology over the past five years have enabled the measurement of single-cells in a multivariate and high-throughput manner. This growth in both dimensionality and quantity of data continues to pose a challenge for effective identification and visualization of rare cell subsets, such as antigen-specific T-cells. Dimension reduction and feature extraction play pivotal role in both identifying and visualizing cell populations of interest in large, multi-dimensional cytometry datasets. However, the automated identification and visualization of rare, high-dimensional cell subsets remains challenging. Here we demonstrate how a systematic and integrated approach combining targeted feature extraction with dimension reduction can be used to identify and visualize biological differences in rare, antigen-specific cell populations. By using OpenCyto to perform semi-automated gating and features extraction of flow cytometry data, followed by dimensionality reduction with t-SNE we are able to identify polyfunctional subpopulations of antigen-specific T-cells and visualize treatment-specific differences between them
Application of fluorescent dye substrates for functional characterization of ABC  multidrug transporters at a single cell level

ABC multidrug transporters are key players in cancer multidrug resistance and in  determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive  and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a  single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. (c) 2016 International Society for Advancement of Cytometry
Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data

Recent technological developments in high-dimensional flow cytometry and mass cytometry (CyTOF) have made it possible to detect expression levels of dozens of protein markers in thousands of cells per second, allowing cell populations to be characterized in unprecedented detail. Traditional data analysis by "manual gating" can be inefficient and unreliable in these high-dimensional settings, which has led to the development of a large number of automated analysis methods. Methods designed for unsupervised analysis use specialized clustering algorithms to detect and define cell populations for further downstream analysis. Here, we have performed an up-to-date, extensible performance comparison of clustering methods for high-dimensional flow and mass cytometry data. We evaluated methods using several publicly available data sets from experiments in immunology, containing both major and rare cell populations, with cell population identities from expert manual gating as the reference standard. Several methods performed well, including FlowSOM, X-shift, PhenoGraph, Rclusterpp, and flowMeans. Among these, FlowSOM had extremely fast runtimes, making this method well-suited for interactive, exploratory analysis of large, high-dimensional data sets on a standard laptop or desktop computer. These results extend previously published comparisons by focusing on high-dimensional data and including new methods developed for CyTOF data. R scripts to reproduce all analyses are available from GitHub (https://github.com/lmweber/cytometry-clustering-comparison), and pre-processed data files are available from FlowRepository (FR-FCM-ZZPH), allowing our comparisons to be extended to include new clustering methods and reference data sets
Guess what: Chronic 13q14.3+/CD5‐ /CD23+ lymphocytic leukemia in blood and t(11;14)(q13;q32)+/CD5+/CD23‐ mantle cell lymphoma in lymph nodes!

Cytometry B Clin Cytom. 2003 Jan;51(1):41-4.

Guess what: Chronic 13q14.3+/CD5-/CD23+ lymphocytic leukemia in blood and t(11;14)(q13;q32)+/CD5+/CD23- mantle cell lymphoma in lymph nodes!
Lima M, Pinto L, Dos Anjos Teixeira M, Canelhas A, Mota A, Cabeda JM, Silva C, Queirós ML, Fonseca S, Santos AH, Brochado P, Justiça B.

Service of Clinical Hematology, Hospital Geral de Santo António, Porto, Portugal. mmc.lima@clix.pt

Abstract
We report a case of a patient with two B-cell lymphoproliferative disorders: CD5(-)/CD23(+) B-cell chronic lymphocytic leukemia and CD5(+)/CD23(-) mantle cell lymphoma. These disorders were diagnosed simultaneously based on flow cytometry, immunohistochemistry, fluorescence in situ hybridization, and polymerase chain reaction-based molecular studies. The B-cell lymphocytic leukemia clone predominated in the blood and bone marrow, whereas the mantle cell clone predominated in lymph nodes.

Copyright 2002 Wiley-Liss, Inc.
PMID: 12500296 [PubMed - indexed for MEDLINE
Multiparameter flow cytometric immunophenotyping of epithelial cells in effusions. Technical improvements and apoptosis measurement.

List of papers. The papers are removed from the thesis due to publisher restrictions.<br><br>
Paper I: Dong HP, Holth A, Berner A, Davidson B, Risberg B. Flow cytometric immunphenotyping of epithelial cancer cells in effusions--technical considerations and pitfalls. Cytometry B Clin Cytom 2007;72:332-343. <a href="http://dx.doi.org/10.1002/cyto.b.20172">doi:10.1002/cyto.b.20172</a><br><br>
Paper II: Dong HP, Kleinberg L, Silins I, Flørenes VA, Tropé CG, Risberg B, Nesland JM, Davidson B. Death receptor expression is associated with poor response to chemotherapy and shorter survival in metastatic ovarian carcinoma. Cancer 2008;112:84-93. <a href="http://dx.doi.org/10.1002/cncr.23140">doi:10.1002/cncr.23140</a><br><br>
Paper III: Dong HP, Kleinberg L, Davidson B, Risberg B. Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry. Nat Protoc 2008;3:955-964. <a href="http://dx.doi.org/10.1038/nprot.2008.77">doi:10.1038/nprot.2008.77</a><br><br>
Paper IV: Dong HP, Holth A, Kleinberg L, Ruud MG, Elstrand MB, Tropé CG, Davidson B, Risberg B. Evaluation of cell surface expression of phosphatidylserine in ovarian carcinoma effusions using the annexin-V/7-AAD assay: clinical relevance and comparison with other apoptosis parameters. Am J Clin Pathol 2009;132:756-762. <a href="http://dx.doi.org/10.1309/AJCPAVFA8J3KHPRS">doi:10.1309/AJCPAVFA8J3KHPRS</a><br><br>
Paper V: Dong HP, Holth A, Ruud MG, Emilsen E, Risberg B, Davidson B. Measurement of apoptosis in cytological specimens by flow cytometry: comparison of Annexin V, caspase cleavage and dUTP incorporation assays. Cytopathology 2011;22:365-372. <a href="http://dx.doi.org/10.1111/j.1365-2303.2010.00811.x">doi:10.1111/j.1365-2303.2010.00811.x</a><br><br>
Paper VI: Dong HP, Ree Rosnes AK, Bock AJ, Holth A, Flørenes VA, Trope' CG, Risberg B, Davidson B. Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions. Cytopathology 2011;22:373-382. <a href="http://dx.doi.org/10.1111/j.1365-2303.2011.00929.x">doi:10.1111/j.1365-2303.2011.00929.x</a
Relationships between optical aggregometry (type Born) and flow cytometry in evaluating ADP-induced platelet activation

Background: Platelet response to activating agents is used to monitor the efficacy of anti-aggregation therapies. The aim of our study has been to demonstrate the existence of relationships between early events of ADP-induced platelet activation, measured by flow cytometry and platelet-rich plasma aggregation,quantified by optical aggregometry. Methods: We evaluated peripheral blood of 12 donors. The following parameters were quantified by cytometry after stimulation with adenosine diphosphate (ADP) (0.5, 1, 2, 5, 10, 20 uM): CD62P (P-selectin) and PAC-1 expression, and cytosolic Ca2+ mobilization. Aggregation was measured by optical aggregometry. We also studied 13 patients, undergoing coronary stenting, treated with aspirin (before procedure) or with aspirin plus clopidogrel (after procedure). We evaluated CD62P and PAC-1 expression, aggregation,and vasodilator-stimulated phopshoprotein phosphorylation (platelet reactivity index, PRI). Results: Flow procedures were more sensitive than aggregometry, with a lowest interindividual variability. Linear relationships existed in donors between CD62P expression and Ca2+ mobilization (P<0.0001),and between aggregation and Ca2+ mobilization (P<0.0001). Linear relationships existed between aggregation and CD62P expression, as percentage (P<0.0001), or relative fluorescence intensity (RFI) (P<0.0001). Exponential equations related aggregation and PAC-1 expression, as percentage (P<0.0001), or RFI (P<0.0001). Linear relationships between aggregation and CD62P expression (as percentage) existed in the patients before (P=0.0022) and after procedure (P=0.0020. Exponential relationships between aggregation and PAC-1 expression (as percentage) existed before (P=0.0012) and after procedure (P=0.0024). Linear correlations related aggregation response predicted on CD62P expression, and measured aggregation inhibition after clopidogrel (P=0.0013) as well as predicted aggregation and PRI inhibition (P=0.0031). Conclusions: Tight relationships between aggregation and cytometric quantification of platelet markers in whole blood, in particular CD62P, allow to predict aggregation response to ADP from flow data in patients treated with aspirin alone or with aspirin plus clopidogrel
Variability of CD3 membrane expression and T cell activation capacity

αβT cells have a wide distribution of their CD3 membrane density. The aim of this paper was to evaluate the significance of the CD3 differential expression on T cell subsets. Analysis was performed on healthy donors and renal transplant patients by flowcytometry The results obtained are : 1-CD3 expression was widely distributed (CV =38.3±3.1 to (43±2.3%). 2-The CD4, CD8,CD45 and forward scatter were similarly distributed. 3-The diversity of CD3 expression was direcly related to the clonotypes: γ9, non γ9 from γδT cells and Vβ clonotype from αβT cells (e.g.: Vβ3FITC 7980±1628 Vβ8PE: Vβ20-FITC 11768±1510). 4-Using a computer simulation, we could confirm differential kinetics of T cell activation according to the initial parameters. Finally, in vitro activation was significantly higher on Vβ8 and Vβ9 (high CD3) compared to Vβ2 and Vβ3 (low CD3, P=0.040 to 0.0003). In conclusion: T cells have highly heterogeneous CD3 expression, possibly predetermined and with clear functional significance