Figure 3.
Functional assay of neutrophil-like cells in vitro. (A) Immunocytochemistry of primary (MPO and ELANE), secondary (LTF), and tertiary (MMP9) granules in NeuPs-XL 2 days after removing doxycycline (Dox-off day2), NeuCs-XL, and human primary neutrophils. Scale bar, 10 μm. (B-C) Migration assay of NeuCs-XL and human neutrophils, using a transwell system with 0.1%, 1%, and 10% fetal bovine serum (FBS) or without FBS (B) and with 50 nM LTB4, C5a, and interleukin-8 or without (C). Means of 3 to 6 independent experiments. Statistical significance was calculated using analysis of variance (ANOVA). *P < .05, **P < .01, ***P < .001, ****P < .0001. (D) The proportion of phagocytosis-positive NeuPs-XL, NeuCs-XL, and human neutrophils incubated with S aureus or E coli. Means of 3 independent experiments. Statistical significance was calculated using ANOVA. **P < .01, ****P < .0001. (E) Representative flow cytometry of phagocytosis assay in NeuCs-XL with or without FITC-labeled E coli. (F) Phagocytosis assay in NeuPs-XL, NeuCs-XL, and human neutrophils. Means of 3 independent experiments. Statistical significance was calculated using ANOVA. **P < .01, ****P < .0001. (G) Representative flow cytometry of oxidative burst assay in NeuPs-XL, NeuCs-XL, and human neutrophils with or without phorbol myristate acetate (PMA) stimulation, using dihydrorhodamine 123. (H) Oxidative burst assay in NeuPs-XL, NeuCs-XL, and human neutrophils using dihydrorhodamine 123. Means of 3 or 4 independent experiments. Statistical significance was calculated using ANOVA. ***P < .001, ****P < .0001. (I) Neutrophil extracellular trap formation of NeuCs-XL and human neutrophils without PMA stimulation or after 4-hour PMA stimulation. Scale bar, 10 μm.

Functional assay of neutrophil-like cells in vitro. (A) Immunocytochemistry of primary (MPO and ELANE), secondary (LTF), and tertiary (MMP9) granules in NeuPs-XL 2 days after removing doxycycline (Dox-off day2), NeuCs-XL, and human primary neutrophils. Scale bar, 10 μm. (B-C) Migration assay of NeuCs-XL and human neutrophils, using a transwell system with 0.1%, 1%, and 10% fetal bovine serum (FBS) or without FBS (B) and with 50 nM LTB4, C5a, and interleukin-8 or without (C). Means of 3 to 6 independent experiments. Statistical significance was calculated using analysis of variance (ANOVA). *P < .05, **P < .01, ***P < .001, ****P < .0001. (D) The proportion of phagocytosis-positive NeuPs-XL, NeuCs-XL, and human neutrophils incubated with S aureus or E coli. Means of 3 independent experiments. Statistical significance was calculated using ANOVA. **P < .01, ****P < .0001. (E) Representative flow cytometry of phagocytosis assay in NeuCs-XL with or without FITC-labeled E coli. (F) Phagocytosis assay in NeuPs-XL, NeuCs-XL, and human neutrophils. Means of 3 independent experiments. Statistical significance was calculated using ANOVA. **P < .01, ****P < .0001. (G) Representative flow cytometry of oxidative burst assay in NeuPs-XL, NeuCs-XL, and human neutrophils with or without phorbol myristate acetate (PMA) stimulation, using dihydrorhodamine 123. (H) Oxidative burst assay in NeuPs-XL, NeuCs-XL, and human neutrophils using dihydrorhodamine 123. Means of 3 or 4 independent experiments. Statistical significance was calculated using ANOVA. ***P < .001, ****P < .0001. (I) Neutrophil extracellular trap formation of NeuCs-XL and human neutrophils without PMA stimulation or after 4-hour PMA stimulation. Scale bar, 10 μm.

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