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. Author manuscript; available in PMC: 2013 Jan 1.
Published in final edited form as: J Immunol. 2012 Jun 1;189(1):381–392. doi: 10.4049/jimmunol.1200409

Figure 3. Neutrophil proteases are associated with neutrophil extracellular traps and neuronal death in vitro.

Figure 3

(a) Intracellular PL2-3 staining is significantly reduced in (ii) transmigrated (TM) neutrophils compared to (i) naïve neutrophils (DAPI and PL2-3) (scale bar, 5 μm), (iii) graph showing the percentage of neutrophils present with intracellular PL2-3 staining. (b) Neutrophil-derived mitochondrial DNA is increased in conditioned medium from transmigrated neutrophils but not in that of naïve neutrophils as determined by PCR. (i) mitochondrial and (ii) nuclear DNA was observed in the conditioned medium of transmigrated neutrophils compared to naïve controls. (c) Elevated levels of extracelluar histone-DNA was detected in conditioned medium of naïve neutrophils and transmigrated neutrophils, (d) Neurons were incubated for 2 h and stained with PGP (green), neutrophils were stained with SJC (red) and NETs stained with PL2-3 (blue) (scale bar, 10 μm). (iii) Reduction in neurotoxicity of transmigrated neutrophils in the presence of PL2-3 (15 μg/ml) is seen in neuronal cultures stained with PGP (green) and MAP-2 (red), as compared to the (iv) isotype treated control (scale bars, 30 μm). Maintained structural integrity is shown in cultured neurons (white arrows) after (ii) exposure to transmigrated neutrophils treated with PL2-3 (15 μg/ml) in comparison to (i) neutrophils transmigrated across activated endothelium in the presence of an isotype control (scale bar, 10 μm). (e) Percentage of maintained integrity of neuronal cell bodies after exposure to transmigrated neutrophils treated with PL2-3 (15 μg/ml) in comparison to isotype treated control.(f) Presence of PL2-3 antibody (15 μg/ml) does not affect neutrophil migration across IL-1β-activated brain endothelium after 24 h of migration. Bars in graphs represent the mean +/− SEM from a minimum of 3 independent experiments carried out on separate cultures. *P<0.05 **P<0.01 (Student’s t-test or one-way ANOVA, with a Bonferroni post hoc test or Student’s t-test where appropriate).