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. Author manuscript; available in PMC: 2019 Mar 1.
Published in final edited form as: Arthritis Rheumatol. 2018 Jan 23;70(3):468–474. doi: 10.1002/art.40372

Figure 2.

Figure 2

Mechanisms of NET formation. A, Quantification of NET formation after exposing drug-induced NETs to diphenyleneiodonium chloride (DPI) or to Cl-amidine (peptidylarginine deiminase inhibitor). B, Measurement of calcium flux using Indo-1-AM. Neutrophils from donors were treated in calcium-free media with drug compound [thapsigargin 0.5μM, hydralazine 10μM, or procainamide 40μM]. Calcium levels were quantified as the ratio of bound violet (BUV 395) versus free blue (DAPI) and compared to background. C, Quantification of NET formation after exposing hydralazine-induced NETs to TMB-8, an intracellular calcium antagonist. D, Human Toll-like receptor (TLR) ligand screening to assess activation of TLR pathways by procainamide or hydralazine. E, Quantification of NET formation after exposing drug-induced NETs to a muscarinic receptor antagonist (atropine) or to specific muscarinic subtype receptors antagonists (Telenzepine (muscarinic type 1 (M1) receptor antagonist), Methoctramine (M2 receptor antagonist), or 4-DAMP (M3 receptor antagonist)). ACH= acetylcholine. Results in A represent 5 independent experiments. Results in B are the average of 4 independent experiments with 3–7 replicates per experiment. Results in C and D represent 5 independent experiments. ** = P < 0.01; *** = P < 0.001 **** = P < 0.0001 by Kruskal-Wallis test with Dunn’s post hoc test or Mann-Whitney U test.