Fig 1.
PRISM chart of the systematic literature review.
Table 1.
Overview of in-literature described in vitro NETs inducers.
MOI: Multiplicity Of Infection (number of bacteria to number of cells). CFU: Colony Forming Units.
Table 2.
Concentrations of the potential NETosis inducers in the experiments.
Fig 2.
NETosis induction for the different inducers.
NETosis was defined as the ratio between the number of Hoechst and PI positive cells. PMA induced NETosis when compared to unstimulated neutrophils, p<0.001 repeated measures ANOVA post-hoc Bonferroni (*) (none n = 5, PMA n = 7, LPS n = 7, glucose n = 5). Error Bars +/- SEM.
Table 3.
Percentage of neutrophils that underwent NETosis.
% NETosis per time point (hr) is given as mean (SEM). P-value of repeated measures ANOVA with Bonferroni post-hoc test results per NETosis inducer versus unstimulated neutrophils.
Fig 3.
NETs formed by S. aureus and E. coli 20 minutes after stimulation for one hour.
DNA (Hoechst, blue, 405) and Extracelullar DNA (PI, red, 561) were stained.
Fig 4.
NETosis per inducer comparing the effect of platelets and the effect of activated platelets supernatant (n = 5, n = 7 respectively).
NETosis was defined as the ratio between Hoechst and PI positive cells. Neutrophils stimulated by platelets were compared against neutrophils stimulated by LPS, platelets + LPS, activated platelets, activated platelets + LPS, activated platelets supernatant, activated platelets supernatant + LPS using repeated measures ANOVA post-hoc Bonferroni (*). No significant differences were found (p>0.05 all). Error Bars +/- SEM.
Fig 5.
The effect of Ionomycin compared to PMA.
(A) Time lapse images of PMA and Ionomycin at different time frames. The arrows indicate the decondensation of the nuclei before (Hoechst, 405) and after (PI, 561) DNA extrusion. (B) The amount of Hoechst and PI positive cells in PMA and Ionomycin stimulated cells and unstimulated cells, show the difference in the process of NETosis. In the PMA stimulated cells, the number of Hoechst positive cells go down as the PI positive cells (NETosis) go up. In the Ionomycin stimulated cells, the number of PI positive cells go up very rapidly, but the Hoechst positive cells remain similar. In unstimulated cells, the intensity of Hoechst staining remains high and no PI staining was detected.