Basic Multicolor Flow Cytometry

Maciorowski, Zofia; Chattopadhyay, Pratip K.; Jain, Paresh

doi:10.1002/cpim.26

Cited by 73 publications

(75 citation statements)

References 46 publications

(50 reference statements)

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“…Data from studies involving multi-centers, each performing its own supervised analysis, have indicated that manual gating introduces the largest variable in standardized experiments (17)(18)(19)(20)(21)40). Data from studies involving multi-centers, each performing its own supervised analysis, have indicated that manual gating introduces the largest variable in standardized experiments (17)(18)(19)(20)(21)40).…”

Section: Discussionmentioning

confidence: 99%

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Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

et al. 2019

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The rapid advancement of immunotherapy strategies has created a need for technologies that can reliably and reproducibly identify rare populations, detect subtle changes in modulatory signals, and assess antigenic expression patterns that are time-sensitive. Accomplishing these tasks requires careful planning and the employment of tools that provide greater sensitivity and specificity without demanding extensive time. Flow Cytometry has earned its place as a preferred analysis platform. This technology offers a flexible path to the interrogation of protein expression patterns and detection of functional properties in cell populations of interest. Mass Cytometry is a newcomer technology that has generated significant interest in the field. By incorporating mass spectrometry analysis to the traditional principles of flow cytometry, this innovative tool promises to significantly expand the ability to detect multiple proteins on a single cell. The use of these technologies in a manner that is consistent and reproducible through multiple sample sets demands careful attention to experiment design, reagent selection, and instrumentation. Whether applying flow or mass cytometry, reaching successful, reliable results involves many factors. Sample preparation, antibody titrations, and appropriate controls are major biological considerations that impact cytometric analysis. Additionally, instrument voltages, lasers, and run quality assessments are essential for ensuring comparability and reproducibility between analyses. In this article, we aim to discuss the critical aspects that impact flow cytometry, and will touch on important considerations for mass cytometry as well. Focusing on their relevance to immunotherapy studies, we will address the importance of appropriate sample processing and will discuss how selection of suitable panels, controls, and antibodies must follow a carefully designed plan. We will also comment on how educated use of instrumentation plays a significant role in the reliability and reproducibility of results.Through this work, we hope to contribute to the effort toward establishing higher standards for rigor and reproducibility of cytometry practices by researchers, operators, and general cytometry users employing cytometry-based assays in their work.

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Section: Discussionmentioning

confidence: 99%

“…Most researchers will begin with the manufacturer recommended titration and test multiple doses until an optimal stain index is achieved (20)(21)(22)(23)(24). Initial titration of antibodies is a wellestablished criterion for good experimental design.…”

Section: Antibody Validationmentioning

confidence: 99%

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

et al. 2019

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“…As a result, critical methods of instrument standardization and calibration (27)(28)(29)(30)(31), panel and experimental design (32)(33)(34)(35)(36), sample preparation and controls (37)(38)(39), spillover correction (40), and data presentation and publication (41,42) are emphasized in various seminal publications. As a result, critical methods of instrument standardization and calibration (27)(28)(29)(30)(31), panel and experimental design (32)(33)(34)(35)(36), sample preparation and controls (37)(38)(39), spillover correction (40), and data presentation and publication (41,42) are emphasized in various seminal publications.…”

Section: Existing Flow Cytometry Resources and Guidelines On Preparinmentioning

confidence: 99%

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments

et al. 2019

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Poor adherence to best practices, insufficient training, and pressure to produce data quickly may lead to publications of suboptimal biomedical research flow cytometry data, which contributes to the body of irreproducible research findings. In addition, documentation of compliance with best flow cytometry practices for submission, visualization, and publication of flow cytometry data is currently endorsed by very few scientific journals, which is particularly concerning as numerous peer-reviewed flow cytometry publications emphasize instrumentation, experimental design, and data analysis as important sources of variability. Guidelines and resources for adequate reporting, annotation and deposition of flow cytometry experiments are provided by MIFlowCyt and the FlowRepository database, and comprehensive expert recommendations covering principles and techniques of field-specific flow cytometry applications have been published. To facilitate the integration of quality-defining parameters into manuscript and grant submission and publication requirements across biomedical fields that rely on the use of flow-cytometry-based techniques, a single comprehensive yet easily and universally applicable document is needed. To produce such a list of gold-standard parameters that assess whether a research flow cytometry experiment has been planned, conducted, interpreted, and reported at the highest standard, a new initiative defining the minimum set of standards a robust and rigorous research flow experiment must fulfill (MiSet RFC Standards) was proposed at CYTO 2019. MiSet RFC Standards will integrate and simplify existing resources to provide a universal benchmark a flow cytometry experiment can easily be measured against. The goal of MiSET RFC Standards is its integration into peerreview and publication procedures through partnership with stakeholders, journals and publishers in biomedical and translational research. This article introduces the aims and anticipated timeline and discusses strategies for interdisciplinary consensus and implementation. A single-resource broadly applicable guideline will harmonize standards across different fields of biomedical research and lead to publication of more robust research findings.

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“…Gating on live T cells (stained with a viability dye and anti-CD4 or anti-CD8), we reproducibly find that 60-95% are GFP+. For details on performing flow cytometry analysis, see Maciorowski, Chattopadhyay, & Jain (2017).…”

Section: Day 2: Repeat Transduction (Optional)mentioning

confidence: 99%

Efficient CRISPR/Cas9‐Mediated Mutagenesis in Primary Murine T Lymphocytes

2018

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The ability to alter gene expression directly in T lymphocytes has provided a powerful tool for understanding T cell biology, signaling, and function. Manipulation of T cell clones and primary T cells has been accomplished primarily through overexpression or gene‐silencing studies using cDNAs or shRNAs, respectively, which are often delivered by retroviral or lentiviral transduction or direct transfection methods. The recent development of CRISPR/Cas9‐based mutagenesis has revolutionized genomic editing, allowing unprecedented genetic manipulation of many cell types with greater precision and ease. This article outlines a protocol for CRISPR/Cas9‐mediated mutagenesis in primary T lymphocytes from Cas9 transgenic mice using retroviral delivery of guide RNAs. © 2018 by John Wiley & Sons, Inc.

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Basic Multicolor Flow Cytometry

Cited by 73 publications

References 46 publications

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

MiSet RFC Standards: Defining a Universal Minimum Set of Standards Required for Reproducibility and Rigor in Research Flow Cytometry Experiments

Efficient CRISPR/Cas9‐Mediated Mutagenesis in Primary Murine T Lymphocytes

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