Additional file 3 of Simplified flow cytometric quantification of human neutrophil extracellular traps (NETs)

Supplementary Fig. 3 Confirmation of absence of neutrophil activation and minimal cytotoxicity using novel HNE-FQ. (A) Quantification of neutrophil CD62L expression with or without HNE-FQ and 100 nM fMLF treatment. n = 3 distinct cell donors. (B) Quantification of neutrophil CD11b expression with or without HNE-FQ and fMLF treatment. n = 3 distinct cell donors. (C) Shape change evaluation of neutrophils untreated or activated with fMLF in the presence or absence of HNE-FQ via quantification of standard deviation from the mean of the forward scatter using flow cytometry. n = 3 distinct cell donors. (D) Representative overlay of forward scatter histograms of untreated (red) or fMLF treated (green) cells. (E) Quantification of HNE-FQ MFI with or without the addition of fMLF. n = 3 distinct cell donors. (F) Quantification of the effect of a range of concentrations of HNE-FQ on cell toxicity after incubation for 3 h using a LDH release assay. 4% Triton-X was used as a positive control. n = 5 distinct cell donors. (G) Representative cytocentrifugation slide of neutrophils after incubation with 5 µM HNE-FQ for 3 h demonstrating healthy cells. Scale = 50 μm. Values are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001