scholarly journals Design and Optimization Protocol for High-Dimensional Immunophenotyping Assays using Spectral Flow Cytometry

2019 ◽  
Author(s):  
L Ferrer-Font ◽  
C Pellefigues ◽  
JU Mayer ◽  
S Small ◽  
MC Jaimes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune System ◽  
Selection Process ◽  
High Dimensional ◽  
Spectral Flow ◽  
High Quality ◽  
Design And Optimization ◽  
Technological Advances ◽  
Large Numbers ◽  
Recent Advancement

ABSTRACTTechnological advances in fluorescence flow cytometry and an ever-expanding understanding of the complexity of the immune system has led to the development of large 20+ flow cytometry panels. Yet, as panel complexity and size increases, so does the difficulty involved in designing a high-quality panel, accessing the instrumentation capable of accommodating large numbers of parameters, and in analysing such high-dimensional data.A recent advancement is spectral flow cytometry, which in contrast to conventional flow cytometry distinguishes the full emission spectrum of each fluorochrome across all lasers, rather than identifying only the peak of emission. Fluorochromes with a similar emission maximum but distinct off-peak signatures can therefore be accommodated within the same flow cytometry panel, allowing greater flexibility in terms of panel design and fluorophore detection.Here, we highlight the specific characteristics regarding spectral flow cytometry and aim to guide users through the process of building, designing and optimising high-dimensional spectral flow cytometry panels using a comprehensive step-by-step protocol. Special considerations are also given for using highly-overlapping dyes and a logical selection process an optimal marker-fluorophore assignment is provided.

Panel Design and Optimization for High‐Dimensional Immunophenotyping Assays Using Spectral Flow Cytometry

2020 ◽  
Vol 92 (1) ◽  
Author(s):  
Laura Ferrer‐Font ◽  
Christophe Pellefigues ◽  
Johannes U. Mayer ◽  
Sam J. Small ◽  
Maria C. Jaimes ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Dimensional ◽  
Spectral Flow ◽  
Design And Optimization ◽  
Panel Design

scholarly journals Acquisition of High‐Quality Spectral Flow Cytometry Data

2020 ◽  
Vol 93 (1) ◽  
Author(s):  
Amy Fox ◽  
Taru S. Dutt ◽  
Burton Karger ◽  
Andrés Obregón‐Henao ◽  
G. Brooke Anderson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Spectral Flow ◽  
High Quality ◽  
Flow Cytometry Data

scholarly journals High‐Dimensional Data Analysis Algorithms Yield Comparable Results for Mass Cytometry and Spectral Flow Cytometry Data

2020 ◽  
Vol 97 (8) ◽  
pp. 824-831 ◽  
Author(s):  
Laura Ferrer‐Font ◽  
Johannes U. Mayer ◽  
Samuel Old ◽  
Ian F. Hermans ◽  
Jonathan Irish ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Data Analysis ◽  
High Dimensional Data ◽  
High Dimensional ◽  
Spectral Flow ◽  
Mass Cytometry ◽  
Flow Cytometry Data ◽  
High Dimensional Data Analysis

scholarly journals With great power comes great responsibility: high-dimensional spectral flow cytometry to support clinical trials

Bioanalysis ◽  
2021 ◽  
Author(s):  
Megan McCausland ◽  
Yi-Dong Lin ◽  
Tania Nevers ◽  
Christopher Groves ◽  
Vilma Decman
Keyword(s):  
Flow Cytometry ◽  
Clinical Trials ◽  
Clinical Sample ◽  
Great Power ◽  
High Dimensional ◽  
Spectral Flow ◽  
Sample Analysis ◽  
Great Responsibility ◽  
Identification And Characterization ◽  
Research Drug

Flow cytometry is a powerful technology used in research, drug development and clinical sample analysis for cell identification and characterization, allowing for the simultaneous interrogation of multiple targets on various cell subsets from limited samples. Recent advancements in instrumentation and fluorochrome availability have resulted in significant increases in the complexity and dimensionality of flow cytometry panels. Though this increase in panel size allows for detection of a broader range of markers and sub-populations, even in restricted biological samples, it also comes with many challenges in panel design, optimization, and downstream data analysis and interpretation. In the current paper we describe the practices we established for development of high-dimensional panels on the Aurora spectral flow cytometer to aid clinical sample analysis.

scholarly journals How to Prepare Spectral Flow Cytometry Datasets for High Dimensional Data Analysis: A Practical Workflow

2021 ◽  
Vol 12 ◽  
Author(s):  
Hannah den Braanker ◽  
Margot Bongenaar ◽  
Erik Lubberts
Keyword(s):  
Flow Cytometry ◽  
Dimensional Analysis ◽  
High Dimensional ◽  
Spectral Flow ◽  
Control Data ◽  
Single Experiment ◽  
Flow Cytometry Data ◽  
Healthy Control ◽  
The Common ◽  
Quality Control Data

Spectral flow cytometry is an upcoming technique that allows for extensive multicolor panels, enabling simultaneous investigation of a large number of cellular parameters in a single experiment. To fully explore the resulting high-dimensional single cell datasets, high-dimensional analysis is needed, as opposed to the common practice of manual gating in conventional flow cytometry. However, preparing spectral flow cytometry data for high-dimensional analysis can be challenging, because of several technical aspects. In this article, we will give insight into the pitfalls of handling spectral flow cytometry datasets. Moreover, we will describe a workflow to properly prepare spectral flow cytometry data for high dimensional analysis and tools for integrating new data at later time points. Using healthy control data as example, we will go through the concepts of quality control, data cleaning, transformation, correcting for batch effects, subsampling, clustering and data integration. This methods article provides an R-based pipeline based on previously published packages, that are readily available to use. Application of our workflow will aid spectral flow cytometry users to obtain valid and reproducible results.

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.

P-067: High dimensional CyTOF analysis of the immune system of multiple myeloma patients with different responses to lenalidomide maintenance

2021 ◽  
Vol 21 ◽  
pp. S75-S76
Author(s):  
Raija Silvennoinen ◽  
Komal Kumar Javarappa ◽  
Sini Luoma ◽  
Philipp Sergeev ◽  
Pekka Anttila ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Immune System ◽  
High Dimensional

scholarly journals Cytotoxicity of Environmentally Relevant Concentrations of Aluminum in Murine Thymocytes and Lymphocytes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Jamal Kamalov ◽  
David O. Carpenter ◽  
Irina Birman
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Immune System ◽  
Dna Binding ◽  
Aluminum Chloride ◽  
Cell Swelling ◽  
Cytotoxic Effects ◽  
Minute Exposure ◽  
Low Concentrations ◽  
Dose Dependent

The effects of low concentrations of aluminum chloride on thymocytes and lymphocytes acutely dissociated from young mice were studied using flow cytometry with a DNA-binding dye. We demonstrate a rapid and dose-dependent injury in murine thymocytes and lymphocytes resulting from exposure to aluminum, as indicated by an increase in the entry into the cell of the DNA-binding dye, propidium iodine. A 60-minute exposure to 10 μM AlCl3caused damage of about 5% of thymocytes, while 50% were injured after 10 minutes at 20 μM. Nearly all thymocytes showed evidence of damage at 30 μM AlCl3after only 5 minutes of incubation. In lymphocytes, injury was observed at 15 μM AlCl3and less than 50% of cells were injured after a 60-minute exposure to 20 μM. Injury only rarely proceeded to rapid cell death and was associated with cell swelling. These results suggest that aluminum has cytotoxic effects on cells of the immune system.

Evolution of mutation rate and virulence among human retroviruses

1994 ◽  
Vol 346 (1317) ◽  
pp. 333-343 ◽  
Keyword(s):  
Immune System ◽  
Mutation Rate ◽  
Hiv Infections ◽  
Molecular Data ◽  
Mutation Rates ◽  
Rapid Progression ◽  
Human T Cell ◽  
Large Numbers ◽  
The Individual ◽  
Multicellular Organisms

High mutation rates are generally considered to be detrimental to the fitness of multicellular organisms because mutations untune finely tuned biological machinery. However, high mutation rates may be favoured by a need to evade an immune system that has been strongly stimulated to recognize those variants that reproduced earlier during the infection, hiv infections conform to this situation because they are characterized by large numbers of viruses that are continually breaking latency and large numbers that are actively replicating throughout a long period of infection. To be transmitted, HIVS are thus generally exposed to an immune system that has been activated to destroy them in response to prior viral replication in the individual. Increases in sexual contact should contribute to this predicament by favouring evolution toward relatively high rates of replication early during infection. Because rapid replication and high mutation rate probably contribute to rapid progression of infections to aids, the interplay of sexual activity, replication rate, and mutation rate helps explain why HIV-1 has only recently caused a lethal pandemic, even though molecular data suggest that it may have been present in humans for more than a century. This interplay also offers an explanation for geographic differences in progression to cancer found among infections due to the other major group of human retroviruses, human T-cell lymphotropic viruses (HTLV). Finally, it suggests ways in which we can use natural selection as a tool to control the aids pandemic and prevent similar pandemics from arising in the future.

Understanding the Islamic State—A Review Essay

2016 ◽  
Vol 40 (4) ◽  
pp. 127-165 ◽  
Author(s):  
Daniel Byman
Keyword(s):  
Review Essay ◽  
Military Force ◽  
Islamic State ◽  
The West ◽  
High Quality ◽  
Terrorist Threat ◽  
Security Forces ◽  
Large Numbers ◽  
Raison D’État ◽  
Foreign Fighters

This article reviews several recent books on the Islamic State in order to understand its goals, motivations, strategy, and vulnerabilities. It argues that the Islamic State's ideology is powerful but also highly instrumental, offering the group legitimacy and recruiting appeal. Raison d'etat often dominates its decisionmaking. The Islamic State's strength is largely a consequence of the policies and weaknesses of its state adversaries. In addition, the group has many weaknesses of its own, notably its brutality, reliance on foreign fighters, and investment in a state as well as its tendency to seek out new enemies. The threat the Islamic State poses is most severe at the local and regional levels. The danger of terrorism to the West is real but mitigated by the Islamic State's continued prioritization of the Muslim world and the heightened focus of Western security forces on the terrorist threat. A high-quality military force could easily defeat Islamic State fighters, but there is no desire to deploy large numbers of Western ground troops, and local forces have repeatedly shown many weaknesses. In the end, containing the Islamic State and making modest rollback efforts may be the best local outcomes.

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