Optimization and standardization of fluorescent cell barcoding for multiplexed flow cytometric phenotyping

Giudice, Valentina; Feng, Xingmin; Young, Neal S.; Biancotto, Angélique

doi:10.1002/cyto.a.23162

Cited by 16 publications

(20 citation statements)

References 22 publications

(65 reference statements)

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“…Fixed cells can also readily be fluorescently barcoded, reducing the total number of tubes, reagents, and potential artificial variability in the data introduced by staining/acquisition on different days. This cutting edge method allows multiplexing of samples individually stained with different concentrations of a N-hydroxysuccinimide dye in one single tube 181920 . Unmixing of the samples is performed by simple gating of each cluster on a dye datagram during data analysis.…”

Section: Discussionmentioning

confidence: 99%

Quantification of airway fibrosis in asthma by flow cytometry

et al. 2018

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Airway fibrosis is a prominent feature of asthma, contributing to the detrimental consequences of the disease. Fibrosis in the airway is the result of collagen deposition in the reticular lamina layer of the subepithelial tissue. Myofibroblasts are the leading cell type involved with this collagen deposition. Established methods of collagen deposition quantification present various issues, most importantly their inability to quantify current collagen biosynthesis occurring in airway myofibroblasts. Here, a novel method to quantify myofibroblast collagen expression in asthmatic lungs is described. Single cell suspensions of lungs harvested from C57BL/6 mice in a standard house dust mite model of asthma were employed to establish a flow cytometric method and compare collagen production in asthmatic and non-asthmatic lungs. Cells found to be CD45 αSMA , indicative of myofibroblasts, were gated, and median fluorescence intensity of the anti-collagen-I antibody labeling the cells was calculated. Lung myofibroblasts with no, medium, or high levels of collagen-I expression were distinguished. In asthmatic animals, collagen-I levels were increased in both medium and high expressers, and the number of myofibroblasts with high collagen-I content was elevated. Our findings determined that quantification of collagen-I deposition in myofibroblastic lung cells by flow cytometry is feasible in mouse models of asthma and indicative of increased collagen-I expression by asthmatic myofibroblasts. © 2018 International Society for Advancement of Cytometry.

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Section: Discussionmentioning

confidence: 99%

Quantification of airway fibrosis in asthma by flow cytometry

et al. 2018

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“…Once barcoded, samples are pooled together for staining and data acquisition on the cytometer, before the pooled data are deconvolved in silico into their original sample identities. Barcoding has been implemented in both flow cytometry 25,26 and mass cytometry. [27][28][29] The difficulty of large studies and data sets Many experiments-typically clinical studies-are simply too large for samples to be processed and run as a single batch, or even at a single site, and require additional measures to decrease the associated variability.…”

Section: The Impact Of Sample Collection and Storage On Experiments Qualitymentioning

confidence: 99%

Section: Step Two: Experimental Designmentioning

confidence: 99%

Making the most of high‐dimensional cytometry data

Marsh‐Wakefield

Mitchell

Norton

et al. 2021

Immunol. Cell Biol.

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High‐dimensional cytometry represents an exciting new era of immunology research, enabling the discovery of new cells and prediction of patient responses to therapy. A plethora of analysis and visualization tools and programs are now available for both new and experienced users; however, the transition from low‐ to high‐dimensional cytometry requires a change in the way users think about experimental design and data analysis. Data from high‐dimensional cytometry experiments are often underutilized, because of both the size of the data and the number of possible combinations of markers, as well as to a lack of understanding of the processes required to generate meaningful data. In this article, we explain the concepts behind designing high‐dimensional cytometry experiments and provide considerations for new and experienced users to design and carry out high‐dimensional experiments to maximize quality data collection.

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“…Another strategy to perform high-content multiplex analysis is based on the fluorescent cell barcoding (FCB) technique [24,25]. In FCB, each sample is labeled with a unique fluorescent signature (barcode), of different fluorescence intensity (due to dilutions) and/or emission wavelength, mixed together with other samples, then stained with antibodies or probes and analyzed by flow cytometry as a single sample.…”

Section: Fluorescent Cell Trackersmentioning

confidence: 99%

Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry

Piwocka¹,

Podszywałow-Bartnicka²,

Swatler³

et al. 2018

Multidimensional Flow Cytometry Techniques for Novel Highly Informative Assays

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Cancer cells, including leukemia cells, reside in a complex microenvironment, which influences biology and activity of the cells. The protective role of bone marrow stromal cells is already commonly recognized. Remodeling of stroma cell functions by leukemia cells is also well documented. In this respect, different routes of interactions were defined, such as direct cell-cell interactions or indirect cross talk, by release of soluble factors or vesicular particles containing proteins, RNAs and other molecules. Since intercellular communication seems to play a role in various biological processes, it might be important to conduct studies in co-culture systems, which at least mimic partially more physiological conditions, and enables this intercellular exchange to occur. Thus, it is crucial to improve analytical methods of investigation of co-cultured cells, to study their interactions and so to understand biology of leukemia in order to understand molecular mechanisms and offer novel therapeutic strategies. The present chapter outlines the importance of modern, multiparameter flow cytometry methods, which allow to analyze interactions between different types of cells within the leukemia microenvironment. Importantly, the proposed experimental setups can be easily transformed to study different cell types and different biological systems.

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Optimization and standardization of fluorescent cell barcoding for multiplexed flow cytometric phenotyping

Cited by 16 publications

References 22 publications

Quantification of airway fibrosis in asthma by flow cytometry

Quantification of airway fibrosis in asthma by flow cytometry

Making the most of high‐dimensional cytometry data

Insight into the Leukemia Microenvironment and Cell-cell Interactions Using Flow Cytometry

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