High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: A potential source of error in analysis of blood samples utilizing fluorescein‐conjugated reagents in flow cytometry

Bedner, Elżbieta; Halicka, H. Dorota; Cheng, Wei; Salomon, T.; Deptała, Andrzej; Gorczyca, Wojciech; Melamed, Myron R.; Darżynkiewicz, Zbigniew

doi:10.1002/(sici)1097-0320(19990501)36:1<77::aid-cyto10>3.3.co;2-6

Cited by 27 publications

(22 citation statements)

References 26 publications

(33 reference statements)

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“…For instance, eosinophils have alkaline cytoplasm and do not express CD16. In this study we used the acidic fluorochrome, fluorescein isothiocynate (FITC) tagged to anti-CD16 antibody to identify PMNs/monocytes/NK cells and observed that the alkaline cytoplasm of eosinophils non-specifically bound to FITC giving the erroneous impression that eosinophils express CD16, as others have previously reported [35]. Thus, care must be taken when trying to identify eosinophils within mixed cell populations using FITC-labelled antibodies and we urge the use of Siglec-8 antibodies to identify bono fide eosinophils, and to avoid such errors.…”

Section: Discussionmentioning

confidence: 83%

Characterisation of Leukocytes in a Human Skin Blister Model of Acute Inflammation and Resolution

et al. 2014

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There is an increasing need to understand the leukocytes and soluble mediators that drive acute inflammation and bring about its resolution in humans. We therefore carried out an extensive characterisation of the cantharidin skin blister model in healthy male volunteers. A novel fluorescence staining protocol was designed and implemented, which facilitated the identification of cell populations by flow cytometry. We observed that at the onset phase, 24 h after blister formation, the predominant cells were CD16hi/CD66b+ PMNs followed by HLA-DR+/CD14+ monocytes/macrophages, CD11c+ and CD141+ dendritic cells as well as Siglec-8+ eosinophils. CD3+ T cells, CD19+ B cells and CD56+ NK cells were also present, but in comparatively fewer numbers. During resolution, 72 h following blister induction, numbers of PMNs declined whilst the numbers of monocyte/macrophages remain unchanged, though they upregulated expression of CD16 and CD163. In contrast, the overall numbers of dendritic cells and Siglec-8+ eosinophils increased. Post hoc analysis of these data revealed that of the inflammatory cytokines measured, TNF-α but not IL-1β or IL-8 correlated with increased PMN numbers at the onset. Volunteers with the greatest PMN infiltration at onset displayed the fastest clearance rates for these cells at resolution. Collectively, these data provide insight into the cells that occupy acute resolving blister in humans, the soluble mediators that may control their influx as well as the phenotype of mononuclear phagocytes that predominate the resolution phase. Further use of this model will improve our understanding of the evolution and resolution of inflammation in humans, how defects in these over-lapping pathways may contribute to the variability in disease longevity/chronicity, and lends itself to the screen of putative anti-inflammatory or pro-resolution therapies.

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Section: Discussionmentioning

confidence: 83%

Characterisation of Leukocytes in a Human Skin Blister Model of Acute Inflammation and Resolution

et al. 2014

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“…While both the genuine apoptotic cells and the “false positive” cells contained numerous DNA strand breaks and were indistinguishable by FCM, the analysis of their morphology by LSC allowed their positive identification (13). In another study, eosinophils were identified by LSC as “false positive” apoptotic cells due to their nonspecific labeling with fluorescein-conjugated reagents (63). LSC was also helpful in distinguishing apoptotic cells from cells infected by Human Granulocytic Erlichiosis (64).…”

Section: The Relocation Attributementioning

confidence: 99%

Laser Scanning Cytometry: Principles and Applications—An Update

Pożarowski

Holden²,

Darżynkiewicz

2012

Methods in Molecular Biology

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Laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of unique analytical capabilities, not provided by flow cytometry (FCM). This review describes attributes of LSC and covers its numerous applications derived from plentitude of the parameters that can be measured. Among many LSC applications the following are emphasized: (a) assessment of chromatin condensation to identify mitotic, apoptotic cells, or senescent cells; (b) detection of nuclear or mitochondrial translocation of critical factors such as NF-κB, p53, or Bax; (c) semi-automatic scoring of micronuclei in mutagenicity assays; (d) analysis of fluorescence in situ hybridization (FISH) and use of the FISH analysis attribute to measure other punctuate fluorescence patterns such as γH2AX foci or receptor clustering; (e) enumeration and morphometry of nucleoli and other cell organelles; (f) analysis of progeny of individual cells in clonogenicity assay; (g) cell immunophenotyping; (h) imaging, visual examination, or sequential analysis using different probes of the same cells upon their relocation; (i) in situ enzyme kinetics, drug uptake, and other time-resolved processes; (j) analysis of tissue section architecture using fluorescent and chromogenic probes; (k) application for hypocellular samples (needle aspirate, spinal fluid, etc.); and (l) other clinical applications. Advantages and limitations of LSC are discussed and compared with FCM.

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“…Since FITC is a non-specific dye, physical absorption of dyes throughout the whole tissue takes place as the main staining mechanism. Nevertheless, it can still be clearly observed that the inner part of each myocyte was stained by more FITC due to its slight binding ability to primary amine groups of proteins 42,43 . The DIC and confocal images of adipose tissue stained with BODIPY are given in Fig.…”

Section: Resultsmentioning

confidence: 97%

Versatile tissue lasers based on high-Q Fabry–Pérot microcavities

Chen

Zhang

et al. 2017

Lab Chip

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Biolasers are an emerging technology for next generation biochemical detection and clinical applications. Progress has recently been made to achieve lasing from biomolecules and single living cells. Tissues, which consist of cells embedded in extracellular matrix, mimic more closely the actual complex biological environment in a living body and therefore are of more practical significance. Here, we developed a highly versatile tissue laser platform, in which tissues stained with fluorophores are sandwiched in a high-Q Fabry-Pérot microcavity. Distinct lasing emissions from muscle and adipose tissues stained respectively with fluorescein isothiocyanate (FITC) and boron-dipyrromethene (BODIPY), and hybrid muscle/adipose tissue with dual-staining were achieved with a threshold of only ~10 μJ/mm2. Additionally, we investigated how tissue structure/geometry, tissue thickness, and staining dye concentration affect the tissue laser. Lasing emission from FITC conjugates (FITC-phalloidin) that target specifically F-actin in muscle tissues was also realized. It is further found that, despite large fluorescence spectral overlap between FITC and BODIPY in tissues, their lasing emissions could be clearly distinguished and controlled due to their narrow lasing bands and different lasing thresholds, thus enabling highly multiplexed detection. Our tissue laser platform can be broadly applicable to various types of tissues/diseases. It provides a new tool for a wide range of biological and biomedical applications, such as diagnostics/screening of tissues and identification/monitoring of biological transformations in tissue engineering.

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High affinity binding of fluorescein isothiocyanate to eosinophils detected by laser scanning cytometry: A potential source of error in analysis of blood samples utilizing fluorescein‐conjugated reagents in flow cytometry

Cited by 27 publications

References 26 publications

Characterisation of Leukocytes in a Human Skin Blister Model of Acute Inflammation and Resolution

Characterisation of Leukocytes in a Human Skin Blister Model of Acute Inflammation and Resolution

Laser Scanning Cytometry: Principles and Applications—An Update

Versatile tissue lasers based on high-Q Fabry–Pérot microcavities

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