![Figure 3. Plasma from TNF-α–stimulated SCD mice and crisis SCD patients induce NET formation in TNF-α–primed neutrophils. (A) Representative immunofluorescence images of NETs released by TNF-α–primed wild-type BM neutrophils. DNA was stained with SYTO13 (cell-permeable nuclear acid dye, green) and Sytox orange (cell-impermeable nuclear acid dye, red). Primed wild-type neutrophils were stimulated with plasma of either TNF-α–treated SA mice (left) or TNF-α–treated SCD mice (right). Scale bar, 10 μm. (B) Representative immunofluorescence images of DNA (green/SYTO13), H3Cit (red/Alexa Fluor 568, i), or NE (red/Alexa Fluor 568, ii) of NETs generated by TNF-α–primed wild-type neutrophils that were stimulated with plasma of TNF-α–treated SCD mice. Insets show NETs stained with DNA dye (green/SYTO13) and isotype control antibodies for H3Cit and NE. Scale bar, 10 μm. (C) Quantification of NETs released by TNF-α–primed wild-type BM neutrophils stimulated with plasma of TNF-α–treated SA mice (black bar) and TNF-α–treated SCD mice (white bar). Results are average values from 4 independent experiments (mean ± SEM, **P < .01). (D) Quantification of NET biomarkers, plasma DNA (left), and nucleosome (right) of non-SCD individuals (gray circle, n = 5), steady-state SCD patients (yellow circle, n = 7), and crisis SCD patients (red circle, n = 10; Mann-Whitney test, median [IQR], *P < .05). (E) Quantification of plasma MPO activity of non-SCD individuals (gray circle, n = 4), steady-state SCD patients (yellow circle, n = 7), and crisis SCD patients (red circle, n = 10; Mann-Whitney test, median [IQR], *P < .05, **P < .01). (F) Representative immunofluorescence images of DNA (green/SYTO13), H3Cit (red/Alexa Fluor 568, i), or NE (red/Alexa Fluor 568, ii) of NETs generated by TNF-α–primed human neutrophils that were stimulated with plasma of crisis SCD patient. Insets show NETs stained with DNA dye (green/SYTO13) and isotype control antibodies for H3Cit and NE. Scale bar, 10 μm. (G) Quantification of NETs released by TNF-α–primed human neutrophils stimulated with plasma of non-SCD individuals (gray circle, n = 3), steady-state SCD patients (yellow circle, n = 7), and crisis SCD patients (red circle, n = 8; mean ± SEM, *P < .05, **P < .01).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/123/24/10.1182_blood-2013-10-529982/4/3818f3.jpeg?Expires=1724199263&Signature=rGx-cA11kGP12Pj-AjzAnn9xFwODNUO~7U-EhdiH5dng-mZmQyfJg60wiLFPGjyjhvcj4Fkdso9pft5~0TXzMbjl4v6wPmCeOI4fxPvjgcF2VmzdpfZR9IcjZBi0t3swNR8z2VJ7ZqgSB~FrFPSXEg7XKb9pck2ayyIWs-GdNMsdMKa7DzbB7Q1jn3FIYPASBjZ-f31tDuHfT5sQuZq9ExR~ZNAO0sM0iGZ8Zgz7xs812VXzC6K9HJEawpP9GAU8waNC~VFOHs5eo0HiCw3IGXrD4rUumvorQ7U6IREwp6KpMlOwqOZ9Bss1by06RvlhDQmMlB1U7pN0ZzUwKIev4g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Plasma from TNF-α–stimulated SCD mice and crisis SCD patients induce NET formation in TNF-α–primed neutrophils. (A) Representative immunofluorescence images of NETs released by TNF-α–primed wild-type BM neutrophils. DNA was stained with SYTO13 (cell-permeable nuclear acid dye, green) and Sytox orange (cell-impermeable nuclear acid dye, red). Primed wild-type neutrophils were stimulated with plasma of either TNF-α–treated SA mice (left) or TNF-α–treated SCD mice (right). Scale bar, 10 μm. (B) Representative immunofluorescence images of DNA (green/SYTO13), H3Cit (red/Alexa Fluor 568, i), or NE (red/Alexa Fluor 568, ii) of NETs generated by TNF-α–primed wild-type neutrophils that were stimulated with plasma of TNF-α–treated SCD mice. Insets show NETs stained with DNA dye (green/SYTO13) and isotype control antibodies for H3Cit and NE. Scale bar, 10 μm. (C) Quantification of NETs released by TNF-α–primed wild-type BM neutrophils stimulated with plasma of TNF-α–treated SA mice (black bar) and TNF-α–treated SCD mice (white bar). Results are average values from 4 independent experiments (mean ± SEM, **P < .01). (D) Quantification of NET biomarkers, plasma DNA (left), and nucleosome (right) of non-SCD individuals (gray circle, n = 5), steady-state SCD patients (yellow circle, n = 7), and crisis SCD patients (red circle, n = 10; Mann-Whitney test, median [IQR], *P < .05). (E) Quantification of plasma MPO activity of non-SCD individuals (gray circle, n = 4), steady-state SCD patients (yellow circle, n = 7), and crisis SCD patients (red circle, n = 10; Mann-Whitney test, median [IQR], *P < .05, **P < .01). (F) Representative immunofluorescence images of DNA (green/SYTO13), H3Cit (red/Alexa Fluor 568, i), or NE (red/Alexa Fluor 568, ii) of NETs generated by TNF-α–primed human neutrophils that were stimulated with plasma of crisis SCD patient. Insets show NETs stained with DNA dye (green/SYTO13) and isotype control antibodies for H3Cit and NE. Scale bar, 10 μm. (G) Quantification of NETs released by TNF-α–primed human neutrophils stimulated with plasma of non-SCD individuals (gray circle, n = 3), steady-state SCD patients (yellow circle, n = 7), and crisis SCD patients (red circle, n = 8; mean ± SEM, *P < .05, **P < .01).
