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. 2019 May 2;20(9):2174. doi: 10.3390/ijms20092174

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© 2019 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Inhibition of PAD-dependent NETs formation and release. (A) Human neutrophils isolated from healthy blood donors were allowed to adhere to coverslips pre-coated with poly-L-lysine. They were pre-incubated with DMSO alone, or with 100 μM of compound 4 or Cl-amidine and then stimulated with PMA. Immunostaining for histone H2 was performed with allophycocyanin (APC) F(ab’)₂ goat anti-rabbit antibodies against histone H2A (red) with DNA counterstained with Hoechst 33258 (blue). Representative immunofluorescent images of 2 independent experiments are shown. (B) Human neutrophils isolated from healthy blood donors were allowed to adhere to a 24-wells plate coated with poly-L-lysine. They were pre-incubated with DMSO alone, or with either 30 μM or 100 μM of compound 4 or Cl-amidine and then stimulated with PMA. Supernatants were collected and quantification of extracellular DNA release was performed with the DNA-intercalating dye, Picogreen. Results are expressed as mean ± SD, with n ≥ 3 and the differences were considered significant if (*) p < 0.05 or (**) p ≤ 0.01. RFU—relative fluorescence units.

Inhibition of PAD-dependent NETs formation and release. (A) Human neutrophils isolated from healthy blood donors were allowed to adhere to coverslips pre-coated with poly-L-lysine. They were pre-incubated with DMSO alone, or with 100 μM of compound 4 or Cl-amidine and then stimulated with PMA. Immunostaining for histone H2 was performed with allophycocyanin (APC) F(ab’)₂ goat anti-rabbit antibodies against histone H2A (red) with DNA counterstained with Hoechst 33258 (blue). Representative immunofluorescent images of 2 independent experiments are shown. (B) Human neutrophils isolated from healthy blood donors were allowed to adhere to a 24-wells plate coated with poly-L-lysine. They were pre-incubated with DMSO alone, or with either 30 μM or 100 μM of compound 4 or Cl-amidine and then stimulated with PMA. Supernatants were collected and quantification of extracellular DNA release was performed with the DNA-intercalating dye, Picogreen. Results are expressed as mean ± SD, with n ≥ 3 and the differences were considered significant if (*) p < 0.05 or (**) p ≤ 0.01. RFU—relative fluorescence units.