Induction of NET formation by gDNA and mtDNA and SCD plasma, with and without BX795. (A) Fluorescence microscopy images from a representative experiment showing NET formation following 7 hours of treatment with a similar concentration of gDNA purified from whole blood or mtDNA purified from platelets or 10% plasma with high cf-mtDNA from a patient with SCD. As a control, neutrophils were left untreated in RPMI 1640 or in RPMI 1640 containing AE Buffer (the buffer in which gDNA and mtDNA were eluted following purification with a QIAGEN kit). Original magnification ×20. (B) NET production (average number of NETs ± SD) induced with AE Buffer (n = 2; 1.67 ± 2.92), gDNA (n = 2; 0.80 ± 1.60), purified mtDNA (n = 5; 14.00 ± 9.70), or cf-mtDNA SCD plasma (n = 5; 18.90 ± 8.80). Representative fluorescence microscopy images (C; original magnification ×20) and NET counts (D) showing that a 4-hour pretreatment with 10 mM BX-795 decreased NET production after a 5 hour-induction with 500 mM purified mtDNA (n = 3; ***P = .0004) or cf-mtDNA in SCD plasma (n = 5; ****P = .0001), but it did not affect the basal NETosis with healthy plasma (H-Plasma; n = 3; P = .1170). n.s., not significant.