![Figure 1. NETs are present within pulmonary vessels of TNF-α–stimulated SCD mice. (A) Representative images of NETs (DNA/red/Sytox orange) detected in the lungs of TNF-α–stimulated SCD mice (i) and the lack of NETs in TNF-α–stimulated SA mice (ii). CD31 (blue/Alexa Fluor 647) labeled the endothelial cells. Scale bar, 10 μm. For imaging, different areas along the dissection were scanned down to an approximate 70 to 80 μm depth using an Axio Examiner.D1 microscope (Zeiss) equipped with a Yokogawa CSU-X1 confocal scan head with a 4-stack laser system (405-nm, 488-nm, 561-nm, and 642-nm wavelengths). Images were obtained using a 20× water immersion objective and as 3-dimensional stacks using Slidebook software (Intelligent Imaging Innovations). (B) Representative histological images of NETs in the lungs of TNF-α–stimulated SCD mice (i,ii, arrows indicate NETs) and the lack of NETs in TNF-α–stimulated SA mice (iii,iv). Scale bar, 10 μm. Images were captured using a Zeiss Axio Observer Z1 microscope equipped with a Zeiss Axiocam HRc camera (color) and a 63× oil immersion objective. (C) Representative immunofluorescence images of the lung sections of TNF-α–stimulated SCD mice showing colocalization of extracellular DNA (green/SYTO 13) and NE (red/Alexa Fluor 568, i,ii) or H3Cit (red/Alexa Fluor 568, iii,iv). CD31 (blue/Alexa Fluor 647) labeled the endothelial cells. Scale bar, 10 μm. Images were captured using an Axio Examiner.D1 microscope equipped with a Yokogawa CSU-X1 confocal scan head with a 4-stack laser system (405-nm, 488-nm, 561-nm, and 642-nm wavelengths) and a 20× water immersion objective. Images were obtained using Slidebook software. (D) Quantification of NETs in the lungs of TNF-α–stimulated SA mice (gray bar, n = 3), SCD mice (white bar, n = 3), and PBS-treated SCD mice (green bar, n = 3). Results presented are the average values (mean ± SEM) of independent experiments with sex- and age-matched mice. *P < .05, ****P < .0001. (E) Quantification of NET biomarkers, plasma DNA (left), and plasma nucleosome (right) of TNF-α–treated SA mice (gray circle, n = 5), SCD mice (white circle, n = 5), and PBS-treated SCD mice (green circle, n = 5; Mann-Whitney test, median [IQR], *P < .05, **P < .01). (F) Quantification of plasma MPO activity of TNF-α–treated SA mice (gray circle, n = 4), SCD mice (white circle, n = 6), and PBS-treated SCD mice (green circle, n = 4; Mann-Whitney test, median [IQR], **P < .01).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/123/24/10.1182_blood-2013-10-529982/4/3818f1.jpeg?Expires=1724198504&Signature=GqJi~US5xuiE~axSgJI2OCtUxLItiSuJmOefOTpAmcKwpFLcqtvJ1zwnjpRBaIPYpr-77T0DesYwXCdenjHxnnI~DSojS95HmHdXDR7yzK~gXZHy-yZaW1l0FHNhHUb~RbaBMNMMcQCBBxkWYoaYnQir-Sd7q3Iqb4tmRxGyaqPHck3-xKD2hFlh-qqVcPMfVCHaUhl41Sbiw5jNd31s1JY8GMVdOus3cceEDKAd8AIVMxj7y0LZFF0ExC3TH3NVZ9tDlt7-pfyUBVXfbXp0oNeSSgd56AOufP8fEPtPBDdokQtf2XXGL~Sg-Yiq6bwoAvrBCEIJd8amJs-KH5YIkg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
NETs are present within pulmonary vessels of TNF-α–stimulated SCD mice. (A) Representative images of NETs (DNA/red/Sytox orange) detected in the lungs of TNF-α–stimulated SCD mice (i) and the lack of NETs in TNF-α–stimulated SA mice (ii). CD31 (blue/Alexa Fluor 647) labeled the endothelial cells. Scale bar, 10 μm. For imaging, different areas along the dissection were scanned down to an approximate 70 to 80 μm depth using an Axio Examiner.D1 microscope (Zeiss) equipped with a Yokogawa CSU-X1 confocal scan head with a 4-stack laser system (405-nm, 488-nm, 561-nm, and 642-nm wavelengths). Images were obtained using a 20× water immersion objective and as 3-dimensional stacks using Slidebook software (Intelligent Imaging Innovations). (B) Representative histological images of NETs in the lungs of TNF-α–stimulated SCD mice (i,ii, arrows indicate NETs) and the lack of NETs in TNF-α–stimulated SA mice (iii,iv). Scale bar, 10 μm. Images were captured using a Zeiss Axio Observer Z1 microscope equipped with a Zeiss Axiocam HRc camera (color) and a 63× oil immersion objective. (C) Representative immunofluorescence images of the lung sections of TNF-α–stimulated SCD mice showing colocalization of extracellular DNA (green/SYTO 13) and NE (red/Alexa Fluor 568, i,ii) or H3Cit (red/Alexa Fluor 568, iii,iv). CD31 (blue/Alexa Fluor 647) labeled the endothelial cells. Scale bar, 10 μm. Images were captured using an Axio Examiner.D1 microscope equipped with a Yokogawa CSU-X1 confocal scan head with a 4-stack laser system (405-nm, 488-nm, 561-nm, and 642-nm wavelengths) and a 20× water immersion objective. Images were obtained using Slidebook software. (D) Quantification of NETs in the lungs of TNF-α–stimulated SA mice (gray bar, n = 3), SCD mice (white bar, n = 3), and PBS-treated SCD mice (green bar, n = 3). Results presented are the average values (mean ± SEM) of independent experiments with sex- and age-matched mice. *P < .05, ****P < .0001. (E) Quantification of NET biomarkers, plasma DNA (left), and plasma nucleosome (right) of TNF-α–treated SA mice (gray circle, n = 5), SCD mice (white circle, n = 5), and PBS-treated SCD mice (green circle, n = 5; Mann-Whitney test, median [IQR], *P < .05, **P < .01). (F) Quantification of plasma MPO activity of TNF-α–treated SA mice (gray circle, n = 4), SCD mice (white circle, n = 6), and PBS-treated SCD mice (green circle, n = 4; Mann-Whitney test, median [IQR], **P < .01).
