Redsafe

Overnight cultures of S. aureus and E. coli were grown in BHI medium and P. aeruginosa in BM2 medium, normalized to an OD₆₀₀ = 1 and supernatants were collected by centrifugation at 8000 rpm for 3 minutes. 15 µL of supernatant was incubated with 5 µg of P. aeruginosa genomic DNA for 1 h at 37°C. Pseudomonas aeruginosa genomic DNA was purified using the Wizard Genomic DNA purification kit (Promega). DNA degradation was visualized on red safe (FroggaBio) stained 1% agarose gels. To test whether exposure to NETs induced DNase production, supernatants from S. aureus, E. coli and P. aeruginosa incubated in HBSS lacking cations with 10⁶ PMA-stimulated human neutrophils (MOI 10:1, same method as described in the NET killing experiments section) were collected by centrifugation at 8000 rpm for 3 minutes. 100 µL of the supernatants were then coincubated at 37°C with 5 µg salmon sperm DNA stained with 2.5 µM Sytox green. 90 kU of DNase I was included as a positive control. Reactions were placed in 96-well black plates with a transparent bottom and Sytox green fluorescence quantified after 1 hour in a Wallac Victor³ luminescence plate reader.

Endpoint RT-PCR was performed on total RNA. First-strand synthesis was performed previously on RNA extracted from edited lines for qPCR (see above). The same cDNA was amplified for 30 cycles using primer pairs P243 and P244 (see Supplementary Dataset 1) to amplify cDNA with/without exon 6. PCR products were separated on 2% agarose gels stained with 0.05 % Redsafe (FroggaBio). Gel bands in the same figure were derived from the same experiment and processed in parallel.

HAPP transgene PCR amplification was performed using Taq DNA polymerase (New England Biolabs, Whitby, ON, Canada) using the following primers: (hAPP) 5′-AACACAGAAAACGAAGTT-3′ and 5′-CCGATGGGTAGTGAAGCA-3′ (480-bp amplicon)^{31 (link)}, (Hypoxanthine-guanine phosphoribosyltransferase, HPRT) 5′-GTAATGATCAGTCAACGGGGGAC-3′ and 5′-CCAGCAAGCTTGCAACCTTAACCA-3′ (177-pb amplicon) (Carcinogenesis). Products were visualized on RedSafe (FroggaBio, North York, ON, Canada) stained 2% agarose gels. Real-time PCR experiments were performed with SYBR Green Taq Mastermix (Quanta BioSciences, Gaithersburg, MD, USA) on an Applied Biosystems 7500 Fast Real-Time PCR system machine (Applied Biosystems, Foster City, CA, USA). Gene amplification was performed with the following primers: (18s) 5′-GTAACCCGTTGAACCCCAT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′; (IDE) 5′- ACTAACCTGGTGGTGAAG-3′ and 5′- GGTCTGGTATGGGAAATG-3′; (Neprilysin) 5′- TCTTGTAAGCAGCCTCAGCC-3′ and 5′- CTCCCCACAGCATTCTCCAT-3′. Results are expressed as fold-induction values normalized to mean HPRT and 18S reference genes using the Pfaffl’s method.