Reagents
2,7-dichlorodihydrofluorescein diacetate (DCF-DA), Diphenyleneiodonium (DPI), 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U0126), SB202190, and zymosan are purchased from Sigma-Aldrich. Sytox Orange nucleic acid stain (Invitrogen), PicoGreen (Invitrogen), Histone H3 antibody (LSC353149; Life Span BioSciences, Inc), Myeloperoxidase (MPO) antibody (Orb16003; Biorbyt), anti-p38 (Bs3566; Bioworld), anti-ERK (Bs3627), anti-pp38 (Cell Signaling Technology Inc, USA), anti-p-ERK (Cell Signaling Technology Inc, USA) were used in this study.
Isolation of bovine neutrophils
Bovine neutrophils were isolated from peripheral blood of healthy dairy cows by bovine PMN (Polymorphonuclear leukocytes) isolation kit (TianJin HaoYang Biological Manufacture CO. China) according to the manufacturer’s instructions. Briefly, blood was diluted 1:1 with cleaning solution, layered on separating solution in centrifugal tubes, and then centrifuged at 700g for 30min. The lower cellular layer was collected, and washed by erythrocyte lysis solution until the cell pellet became white. Finally, isolated neutrophils were re-suspended in RPMI 1640 medium without phenol red, and placed in a incubator until further use.
Cytotoxicity assay by CCK-8 kit
Briefly, neutrophils were seeded into a 96-well plate, confronted with histamine (6.25, 12.5, 25μM) for 2h. Subsequently, 10μL CCK-8 solution was added into each well. After 2h incubation in an incubator, the plate was read by a plate reader at 450nm.
Lactate dehydrogenase (LDH) assay
Neutrophils were seeded into a 96-well plate and confronted with histamine (6.25, 12.5, 25μM) for 2h. The plate was centrifuged at 400g for 5min, and the supernatants were transferred to a new plate. LDH activity was measured by LDH Cytotoxicity Assay kit (Beyotime Biotechnology, China) according to the manufacturer's protocols.
Confocal microscopy analyses
Isolated neutrophils were seeded on glass coverslips pre-treated with poly L-lelysine (0.1 mg/ml) and stimulated with histamine (25μM) for 2h at 37 °C with 5 % CO2. Then, samples were fixed with 4% (w/v) paraformaldehyde for 30min. For immunostaining, paraformldehyde solution was removed, and samples were washed thrice with PBS (Phosphate buffered saline), followed by 1h incubation with blocking buffer 3% BSA (bovine serum albumin). Labeling of specific proteins (histone and MPO) for NETs was performed with the incubation of anti-histone antibody and anti-myeloperoxidase antibody overnight, and secondary antibody goat-anti-rabbit conjugated to Alexa 488 for 2h. For DNA staining, the samples were incubated with 5μM Sytox Orange (dissolved in PBS) for 10 min in dark. Samples were observed and Images were taken using confocal microscope (Olympus FluoView FV1000).
NETs quantification based on PicoGreen® fluorescent dye
In the first set of experiment, we quantified NETs induced by different concentrations of histamine (6.25, 12.5, 25μM). Neutrophils were seeded in a 96-well plate, and then stimulated with histamine for 2h in an incubator at 37 °C with 5 % CO2. Quant-iT™ PicoGreen solution was added into each well, and the fluorescence intensity was measured using an Infiniti M200 fluorescence plate reader (Tecan, Austria).
In the secondary set of experiment, we examined the effects of specific inhibitors on NET formation induced by histamine (25μM) via NETs quantification. Neutrophils were pretreated with NADPH oxidase inhibitor (DPI), the inhibitors of ERK1/2-signaling pathway (U0126) and P38 MAPK-signaling pathway (SB202190) for 30 min in a 96-well plate, and then challenged by histamine for 2h in an incubator with 37 °C and 5 % CO2. After adding PicoGreen solution, the plate was read by an Infiniti M200 fluorescence plate reader (Tecan, Austria) with 485nm length of excitation and 535nm length of emission.
ROS detection
Neutrophils were seeded into a 96-well plate, and stimulated with histamine (6.25, 12.5, 25μM) for 2h in an incubator with 37 °C and 5 % CO2. DCF-DA (10μM) was added to each well, and the plate was incubated for 20min. After thrice washing with PBS, fluorescence intensity was measured by an Infiniti M200 plate reader at 485 nm of excitation and 525 nm of emission.
Western blotting
For protein isolation, neutrophils were confronted with histamine (6.25, 12.5, 25μM) for 2h, and then lysed with M-PER™ mammalian protein extraction reagent (Thermo Fisher Scientific). After centrifugation, the supernatant was collected, and protein concentration was measured by a bicinchoninic acid (BCA) protein assay reagent kit (Pierce).
For western blotting, firstly we separated proteins in samples using gel electrophoresis. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membrane. Next, the membrane was blocked with 3% BSA to prevent any nonspecific binding of antibodies, incubated with primary antibody (anti-p38 monoclonal antibody, 1:1000; anti-phosphor-p38 monoclonal antibody, 1:1000; anti-ERK monoclonal antibody, 1:1000; anti-phosphor-ERK monoclonal antibody, 1:1000) overnight at 4°C, and incubated with HRP-conjugated secondary antibody for 2h at room temperature. In the end, the membrane was detected using enhanced chemiluminescence (ECL) Plus Western Blotting Detection System (ProteinSimple, San Jose, CA, U.S.A.).
Statistical analysis
All Data were illustrated as means ± SEM of at least three biological replicates and two technical replicates. Graphs and statistical analyses were generated by using GrapPadPrism software (v.7.03). One-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests were used. Statistical significance was defined by a p value < 0.05.