A23187

HNE with 81 U/mg of activity was purchased
from SERVA Electrophoresis (Heidelberg, Germany).
3,4,5-trimethoxybenzoic acid 8-(diethylamino)
octyl ester (TMB-8, Sigma-Aldrich,
Canada), A23187 (Santa Cruz Biotechnology,
USA), Rottlerin Tumor necrosis factor-alpha
(TNF-α)protease inhibitor I (TAPI-1), U73122,
R59022 (Merck Millipore, USA), and pyrrolidinedithiocarbamate
(PDTC, BioVision, USA)
were employed to investigate the intracellular
signal transduction pathways involved in IL-10
mRNA expression. The actions of these reagents
are summarized in table 1.
In addition, PKC inhibitors including Ro-
318425 (Merck Millipore), Go 6976, Go 6983,
and CGP 41251 (Tocris Bioscience, UK), as
well as a PKC theta/delta inhibitor (Merck Millipore)
were utilized to investigate the roles of
PKC isoforms in IL-10 production. The PKC
isoform-specific inhibition profile of these reagents
is summarized in table 2.
Xestospongin C, which antagonizes the calciumreleasing
action of IP3 at the receptor level, was
obtained from Sigma-Aldrich, USA. Each reagent
solution was negative for endotoxin according to
the Endospecy test (22 (link), 23 (link)).

Human neutrophils were isolated by centrifuging heparinized venous blood over Histopaque 1119 (Sigma-Aldrich) and subsequently over a discontinuous Percoll (Amersham Biosciences) gradient as previously described (Fuchs et al., 2007 (link)). Experiments were performed in RPMI-1640 (w/o phenol red) supplemented with 10 mM HEPES and 0.05% human serum albumin. Cells were seeded at 10⁵/well (24-well plate) for NET experiments and stimulated with PMA, staurosporine, cycloheximide (Sigma-Aldrich), A23187 (Santa Cruz Biotechnology Inc.), Nigericin (InvivoGen), Candida albicans SC5314 hyphae, GBS, α-hemolysin (generated as previously described [Virreira Winter et al., 2016 (link)]), Staphylococcus aureus (prepared as previously described [Fuchs et al., 2007 (link)]), TNF-α (Thermo Fisher Scientific), z-VAD-FMK (Enzo) or SMAC mimetic (Birinapant, ChemieTek) for 2–6 hr. Where applicable, cells were pre-treated inhibitors for 30 min before stimulation.

Luminol (11050, AAT-Bioquest), horseradish peroxidase (31941, Serva), PMA (P8139, Sigma), A23187 (Santa Cruz Biotechnology Inc), GATA-1 inhibitor (anagrelide, SML0846, Sigma), GATA-2 inhibitor (K-7174, HY-12743A, MedChemExpress) were used. Recombinant murine cytokines (IL-3, IL-5, IL-9, GM-CSF, SCF, G-CSF) were from Peprotech.