Black 96 well plate

To quantify NETs, the method of Parker et al. (11 (link)) was adopted. Briefly, neutrophils (5 × 10⁶ cells/well in a 200 μl volume) were suspended in HBSS (Mediatech-CellGro, USA) and seeded in a black 96-well plate (Cayman Chemical, Ann Arbor, MI, USA). The cells were treated with PMA (1 μg ml⁻¹), P. fluorescens (1 × 10⁶ CFU), V. harveyi (1 × 10⁶ CFU), or E. tarda (1 × 10⁶ CFU) for 1, 2, 3, or 4 h. The control cells were untreated. After treatment, the membrane-impermeable DNA-binding dye, Sytox Green (5 μM), was added to the cells, followed by incubation for 5 min. Fluorescence was then quantified as relative fluorescence units (RFU) at 485 nm excitation and 530 nm emission using a fluorescence spectrophotometer (Infinite M1000, Tecan, Switzerland). For NETs inhibition assay, neutrophils were pre-incubated with the following inhibitors for 30 min at 22°C: 100 μM ROS scavenger Trolox (Sigma, St. Louis, MO, USA), 1 mM nitric oxide (NO) inhibitor N(G)-nitro-L-arginine methylester L-NAME (Sigma, St. Louis, MO, USA), or 100 μM MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH) (Calbiochem, San Diego, CA, USA). NETs production and measurements were then performed as above.

The quantification of ETs was performed as reported previously (Parker et al., 2012 (link)). Briefly, peritoneal cells (1 × 10⁶ cells ml⁻¹) were suspended in L-15 medium and seeded in a black 96-well plate (200 μl well⁻¹) (Cayman Chemical, Ann Arbor, MI, USA). The cells were treated with 500 μg ml⁻¹ of OVA, or 500 μg ml⁻¹ of OVA/Alum for 1, 2, or 4 h. The untreated cells served as control. For the concentration-dependent study of Alum adjuvant, 25, 50, or 100 μg ml⁻¹ of Alum was used to stimulate the cells. After treatment, 10 μl of Sytox Green with 80 mg ml⁻¹ was added to each well, followed by incubation for 5 min. Fluorescence was then quantified as relative fluorescence units (RFU) at 485-nm excitation and 530-nm emission using a fluorescence spectrophotometer (POLARstar OPTIMA, Ortenberg, Germany).