Connected component masking accurately identifies the ratio of phagocytosed and cell‐bound particles in individual cells by imaging flow cytometry

Fei, Chenjie; Lillico, Dustin M.E.; Hall, Brian E.; Rieger, Aja M.; Stafford, James L.

doi:10.1002/cyto.a.23050

Cited by 13 publications

(6 citation statements)

References 18 publications

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“…These issues can be overcome by imaging flow cytometry, where each measured cell is microscopically depicted as well. The application of this technique for phagocytosis was recently illustrated by Fei et al ; however, such instruments are not widely available yet and more expensive than conventional flow cytometers.…”

Section: Discussionmentioning

confidence: 99%

Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry

et al. 2018

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Systemic infections with the opportunistic mold Aspergillus fumigatus are a great threat to immunocompromised patients such as transplant recipients. Immunological research on A. fumigatus involves the measurement of phagocytosis of fungal conidia (spores) by human phagocytes. Here, we present a fast and flexible way to analyze phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC) labeling of conidia prior to co‐incubation with human leukocytes and an anti‐FITC counterstaining step postincubation to allow the discrimination of internalized and adherent conidia. In contrast to many other protocols, this method can be combined with further surface marker analyses. We sought to determine phagocytosis rates of A. fumigatus conidia in different stages and after several incubation times using this method. Moreover, we provide an example of application by comparing phagocytosis of A. fumigatus mutants to the wild type. © 2018 International Society for Advancement of Cytometry

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Section: Discussionmentioning

confidence: 99%

Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry

et al. 2018

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“…After re-suspension, the harvested cells were washed with ice-cold phagocytosis buffer and fixed in 1% paraformaldehyde (PFA, Sigma-Aldrich) prior to analysis using an ImageStream X Mark II instrument (Amnis Corporation). For each sample, 5000 events were collected and data were analyzed using IDEAS ® software (Amnis) to resolve two different phenotypes: i) % of cells with surface-bound beads only, and ii) % of cells with at least one phagocytosed bead, as previously described [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

“…After a 1 h incubation at room temperature, the cells were washed with PBS and re-suspended in 1% PFA prior to analysis using an ImageStream X Mark II instrument. For each sample, 3000 events were collected and phosphotyrosine signals were identified and calculated using the intensity mask feature in the IDEAS ® software as described in [ 27 ].…”

Section: Methodsmentioning

confidence: 99%

“…AD293 cells (2 × 10 5 ) co-expressing 2.6b ITAM CYT and various 1.1b CYT constructs (i.e., 1.1b WT CYT , 1.1b 6YF CYT , 1.1b Prox CYT , 1.1b Distal CYT, and 1.1b 3YF CYT ) were incubated with 4.5 µm yellow-green beads (6 × 10 5 ) opsonized with α-HA and α-FLAG mAbs for 15 min ( A ) and 30 min ( B ) at 37 °C prior to the analysis of samples using the ImageStream X Mark II instrument. Cells with only surface-bound (SB) targets (light grey bars) or with ≥1 phagocytosed (P) bead (black bars) were identified and gated as reported [ 27 ]. The percentage of cells displaying the two phenotypes were calculated as: % cells with (P) bead or % cells with (SB) beads only / % total cells associated with beads.…”

Section: Figurementioning

confidence: 99%

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A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response

Fei

Zwozdesky

Stafford

2020

IJMS

Self Cite

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Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are a family of immunoregulatory proteins shown to regulate several innate immune cell effector responses, including phagocytosis. The precise mechanisms of IpLITR-mediated regulation of the phagocytic process are not entirely understood, but we have previously shown that different IpLITR-types use classical as well as novel pathways for controlling immune cell-mediated target engulfment. To date, all functional assessments of IpLITR-mediated regulatory actions have focused on the independent characterization of select IpLITR-types in transfected cells. As members of the immunoglobulin superfamily, many IpLITRs share similar extracellular Ig-like domains, thus it is possible that various IpLITR actions are influenced by cross-talk mechanisms between different IpLITR-types; analogous to the paired innate receptor paradigm in mammals. Here, we describe in detail the co-expression of different IpLITR-types in the human embryonic AD293 cell line and examination of their receptor cross-talk mechanisms during the regulation of the phagocytic response using imaging flow cytometry, confocal microscopy, and immunoprecipitation protocols. Overall, our data provides interesting new insights into the integrated control of phagocytosis via the antagonistic networking of independent IpLITR-types that requires the selective recruitment of inhibitory signaling molecules for the initiation and sustained cross-inhibition of phagocytosis.

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“…However, the images are 2D rather than 3D, and the assessment of nuclear localization is still mostly qualitative since the calculation of signals within a nuclear mask does not fully account for the 3D depth of the cell, including regions in front of, behind or immediately peripheral to the nucleus, such as the ER . Still, imaging flow cytometry enables a variety of high‐throughput morphological and subcellular localization studies that cannot be done by traditional flow cytometry, and both the technology and masking algorithms are continually improving . Similarly, another technology that enables the assessment of morphological characteristics and subcellular localization is laser‐scanning cytometry (LSC).…”

Section: Introductionmentioning

confidence: 99%

A rapid method for quantifying cytoplasmic versus nuclear localization in endogenous peripheral blood leukocytes by conventional flow cytometry

Brittain

Gulnik

2017

Cytometry Pt A

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A biochemical system and method have been developed to enable the quantitative measurement of cytoplasmic versus nuclear localization within cells in whole blood. Compared with the analyses of nuclear localization by western blot or fluorescence microscopy, this system saves a lot of time and resources by eliminating the necessity of purification and culturing steps, and generates data that are free from the errors and artifacts associated with using tumor cell lines or calculating nuclear signals from 2D images. This user‐friendly system enables the analysis of cell signaling within peripheral blood cells in their endogenous environment, including measuring the kinetics of nuclear translocation for transcription factors without requiring protein modifications. We first demonstrated the efficiency and specificity of this system for targeting nuclear epitopes, and verified the results by fluorescence microscopy. Next, the power of the technique to analyze LPS‐induced signaling in peripheral blood monocytes was demonstrated. Finally, both FoxP3 localization and IL‐2‐induced STAT5 signaling in regulatory T cells were analyzed. We conclude that this system can be a useful tool for enabling multidimensional molecular‐biological analyses of cell signaling within endogenous peripheral blood cells by conventional flow cytometry. © 2017 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC.

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Connected component masking accurately identifies the ratio of phagocytosed and cell‐bound particles in individual cells by imaging flow cytometry

Cited by 13 publications

References 18 publications

Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry

Fast and Quantitative Evaluation of Human Leukocyte Interaction with Aspergillus fumigatus Conidia by Flow Cytometry

A Fish Leukocyte Immune-Type Receptor Uses a Novel Intracytoplasmic Tail Networking Mechanism to Cross-Inhibit the Phagocytic Response

A rapid method for quantifying cytoplasmic versus nuclear localization in endogenous peripheral blood leukocytes by conventional flow cytometry

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