Neutrophil Functional Assays Optimization

Human and mouse neutrophils were seeded on 12-mm glass cover slips in X-VIVO^TM 15 medium (Lonza, Walkersville, MD, USA), primed with 25 ng per ml GM-CSF for 20 min, and subsequently stimulated with 10⁻⁸ M C5a, 100 ng per ml LPS or co-cultured with Escherichia (E.) coli GFP M655 (E. coli-GFP; a kind gift of E. Slack, ETH Zurich) for 15 min. In selected experiments, the following inhibitors were used 30 min before GM-CSF priming: Oligomycin A (2.5, 5, or 10 µg per ml), 2-DG (1, 3, 5 mM), rotenone (10 µM), antimycin A (5 µg per ml), Q-VD (20 µM), taxol (1 µM) and nocodazole (5 µM). In other experiments, cells were pre-cultured for 30 min in the presence of ATP (10, 100 µM, or 1 mM) or NMN (500 μM) and then stimulated for 45 min, besides GM-CSF/C5a, also with RNP-ICs-SLE [IgG, purified from with SLE antibody positive human plasma (Cat # DA1805, Trina Bioreactive AG, Nänikon, Switzerland, dilution 1:33), mixed with small nuclear ribonucleoprotein (SmRNP) (Cat # ATR01-02, Arotec, New Zealand, dilution 1:100)] or RNP-ICs-Ab [an anti-damaged DNA/RNA monoclonal antibody (clone 15A3, Cat # SMC-155, StressMarq Biosciences, Victoria, Canada, dilution 1:33) mixed with SmRNP]^{29 (link)} or P. aeruginosa (ATCC-BAA-47; strain HER-1018)^{42 (link)}; with a multiplicity of infection of 10:1 (bacteria to neutrophils).