D. discoideum and bacteria culture

The D. discoideum laboratory strains AX2 (Ax2–214) and AX4 (Ax4(Ku)33 have been deposited at and can be obtained from the Dicty Stock Center (http://dictybase.org/StockCenter/StockCenter.html), the derived mutant strains AX2 NoxABC-KO and AX4 TirA-KO were generated and are kept in the laboratories of TS and AK, respectively. All D. discoideum strains were cultured axenically in HL-5 medium (Formedium) supplemented with 50 U ml−1 penicillin and 50 μg ml−1 streptomycin (Pen/Strep) at 22 °C in 10 cm Petri dishes. The exponentially growing cells at about 80% confluence were harvested for experiments. To obtain higher cell numbers, D. discoideum was transferred into shaking flasks with HL-5 medium (plus Pen/Strep) at 22 °C, and cells were harvested before the density reached 5 × 106 per ml (ref. 34). The avirulent laboratory bacterial strain of K.p.35 was cultured in SM medium without the antibiotic. The Kp–GFP was kindly provided by Dr P. Cosson (University of Geneva), and cultured at 37 °C in SM medium with 100 μg ml−1 of ampicillin.

Development of D. discoideum slugs

As the experiment requires, D. discoideum slugs can be developed from axenic culture or from a K.p. bacterial lawn. To remove the nutrient medium, D. discoideum axenic cultures were centrifuged twice at low speed and re-suspended in Sorensen buffer. The cell pellet was then plated in a single line on a 1% water agar plate, with dyes (PI or Lucifer Yellow) added while pouring the plate15. Alternatively, K.p. cultured from SM medium were spread on SM agar plates to generate a K.p. lawn after overnight culture at 37 °C. D. discoideum cells were then added on the K.p. bacteria lawns and incubated at 22 °C until phagocytic plaques appeared. The actively replicating cells at the rim of plaques were gently collected with a sterile toothpick and spotted on 1% water agar plates. To increase the efficiency of slug formation, multiple inocula can be spotted on each plate36. The plates for both methods were mildly dried under a hood for 5–10 min and covered with aluminium folio leaving a 2-mm hole on the side opposite the lines of cells to generate a unidirectional light source. Finally, the plates were placed in a humid box at 22 °C with constant lighting. The migrating slugs usually appear 18 h post incubation and remain as long as 48 h. Of note, it is easier to generate slugs from AX2 and strains derived thereof, using the ‘bacteria-toothpick’ method than the axenic development method.

Disaggregation of slugs

Slugs that migrated more than 1 cm from the original spot were carefully collected with a pipette tip and transferred into a 1.5 ml Eppendorf tube containing 1 ml of Sorensen buffer supplemented with 1 mM EDTA37. Up to 15 or 20 slugs can be transferred into 1 tube to increase the cell density. The slugs were incubated in buffer for about 10 min, and then disaggregated by gentle shaking. The disaggregated slug cells were pelleted by quick spin and re-suspended in Sorensen buffer for later use.

FACS sorting of S cells

Slugs were developed on agar plate containing Ethidium Bromide (EB) or other fluid phase dyes, and disaggregated as described above. Then, the S cells stained by EB or other fluid phase dyes, were purified by fluorescence-activated cell sorting (BD FACSAria), as described previously15. The top 1% fluorescent cells (S cells) and the rest of the cell population were collected separately in tubes containing ice-cold Sorensen buffer for later use. The purified S and non-S cells were then exposed to K.p. bacteria or 5 μg ml−1 of K.p. LPS for 2 h followed by PI or SYTO9 staining to visualize DNA.

Cell viability and mtDNA analysis

S cells and non-S cells were purified and exposed for 2 h to 5 μg ml−1 of K.p. LPS, or the combination of LPS and K.p. (multiplicity of infection=50:1). Two hundred cells from each group were then plated on K.p. lawns. The number of viable amoebae cells was determined by counting plaques formed on K.p. lawns after 7 days.

Purified S cells were exposed to 5 μg ml−1 of K.p. LPS for 3 h, and then the medium and cells from culture plates were collected into 1.5 ml centrifuge tubes. The supernatant and the cells were separated by centrifugation (5 min at 1,200g), and the cell pellet was reserved for DNA preparation. Additional centrifugation (5 min, 4,500g) was applied to remove all debris and floating cells from the supernatant. The final supernatant contained mainly ETs. This was confirmed by staining an aliquot with SYBR Green and examining it by phase contrast and fluorescence microscopy. DNA was prepared by the HotSHOT method38 from the final supernatant fraction, and from untreated S cells as a control. The quantitative PCR was performed as described15 using primers (listed in Supplementary Table 1) targeting the mtDNA-encoded rnlA gene, a nuclear-encoded gene histone H3a and any of the 40 nuclear-encoded actin genes. The fold enrichment of mtDNA versus nuclear DNA in the LSP-stimulated S cell pellet and the supernatant fraction were calculated using the ΔΔCt method, by comparison to the non-stimulated S cell pellet as a control. The s.d. of three replicates were calculated and displayed following the Applied Biosystems’ guideline for Real Time Quantitative PCR as the range of variation.

Visualization of ETs

ET visualization can be performed on either disaggregated slug cells or FACS-purified S cells. In disaggregated slug cells, 2.5–20 μg ml−1 of LPS from Klebsiella pneumoniae (Cat: L1519, Sigma) or Pseudomonas aeruginosa (P.a.) (Cat: L9143, Sigma) was added to stimulate ETs formation. About 0.5 μg ml−1 of PI or 5 μM of SYTO9 was added to visualize ETs. Green fluorescent latex beads (Cat:15702, Polysciences) or Kp–GFP (washed twice in Sorensen prior to use) can be added before observation by time-lapse microscopy (ImageXpress Micro XL, Molecular Devices) or standard epifluorescence microscopy (Axiovert 135, Zeiss). Videos were recorded using white light, FITC and DsRed filters with a minimum exposure time, every 5 min for at least 4 h with temperature control at 22 °C.

Alternatively, to directly visualize the fate of mtDNA during ET generation, vegetatively growing cells were incubated with 5 μM MitoSox Red (a mitotropic DNA dye, Cat: M36008, Life Technologies) for 30 min. After removal of excess dye, vegetative cells were developed into slugs. Finally, the slugs were disaggregated and incubated with K.p. LPS for 2 h in dishes before ET imaging. To visualize the fate of mitochondrial proteins from S cells, disaggregated slug cells were incubated with 0.1 μM MitoTracker Green (a mitotropic thiol cross linker dye Cat: M-7514, Life Technologies) for 30 min. After removal of excess dye, the disaggregated slug cells were incubated for an additional 2 h with K.p. LPS in dishes before ET imaging.

Measurement of bacteria viability

The live and dead K.p. bacteria were visualized by using the BacLight bacterial viability assay kit (Invitrogen). All bacteria are stained by SYTO9 and dead bacteria by PI. S cells from AX4 slugs were purified and mixed with K.p. at a 5:1 ratio of bacteria to amoebae to stimulate ET production. The live and dead bacteria in the floating suspension or attached to the ETs were visualized by SYTO9 and PI, respectively. The bacteria were judged to be associated with ETs if their fluorescent signal overlapped the fluorescence from the ET DNA stain. Bacteria not associated with ETs were judged to be in floating suspension, but we could not exclude the possibility that they were associated with an ET fragment, or that they had been associated with an ET at some previous time. In microplate reader, the viability of Kp–GFP was measured by fluorescence intensity of the GFP signal at 400 nm excitation and 512 nm emission.

Visualization of S cells and ROS

S cells were visualized in confocal microscopy (LSM700, Zeiss) by spraying 5 μg ml−1 AO (Cat: A1301, Life Technologies) on slugs or by allowing slugs to migrate on water agar plates containing 2 μg ml−1 Lucifer Yellow (Cat: L0259, Sigma). In situ superoxide production in slugs was visualized by spraying 30 μM DHE (Cat: 37291, Sigma) on slugs with 30-min incubation in the dark before observation by confocal microscopy. Of note, to avoid auto-oxidation, the DHE working solution is always freshly prepared before each experiment, and the DHE stock solution is kept in the dark at −20 °C for <10 days after dissolving the powder. Usually, a × 5 or × 10 magnification fits the slug scale with optimal resolution. The object-based 3D reconstruction from stacks of confocal sections and the spatial analysis of pixel intensity were performed with the Imaris software. The results are plotted as 2D histograms of the Lucifer Yellow and DHE channels, allowing to calculate a Pearson’s coefficient for the degree of colocalization.

To compare the number and location of foci of in situ ROS production in slugs from different strains, DHE was gently sprayed on slugs and imaged as described above. The 3D reconstruction of each confocal stack was automatically performed by the ImageJ software (see Supplementary Movie 6). Because of the three-dimensionality of the data set, it turned out that the easiest and optimal method was to rely on blind counting by three different lab members of the number of red foci in each slug, while rotating the 3D objects back and forth.

OBG-coated beads were generated to visualize phagosomal ROS generation from S cells. As described20, OBG (Cat: O-13291, Life Technologies) and Alexa fluor 594 (Cat:A20004, Life Technologies) were covalently linked to BSA-coated 3 μm carboxylated silica beads (PSi-3.0COOH, Kisker Biotech), and fed to disaggregated slug cells. During phagocytosis, the ROS-insensitive dye Alexa fluor 594 exhibits stable red fluorescence, while the ROS-sensitive dye OBG changes to green fluorescence due to the oxidation by phagosomal ROS, therefore giving the beads a yellow colour. The videos were recorded as described above.

Quantification of ROS and ETs

Extracellular H 2 O 2 production from disaggregated slug cells was quantified by membrane-impermeant AUR as described20. Disaggregated slug cells suspended in Sorensen buffer were added to a 96-well plate (Cat: 236108, Nunc) at 2 × 105 cells per well, followed by addition of AUR and HRP (Cat: 10108090001, Roche) to the final concentration of 6.25 μM and 0.005 U ml−1, respectively. The volume of each well was brought to 100 μl with Sorensen buffer. About 20 μg ml−1 of P.a. LPS and/or various concentrations of catalase (Cat: C9322, Sigma), as indicated, were added to selected wells before detection. The H 2 O 2 -oxidized AUR was specifically recorded at 530 nm excitation and 590 nm emission from each well, every 2 min for 1.5 h in a microplate reader (Synergy Mx, BioTek) with temperature control at 22 °C. The slopes (relative fluorescence units (r.f.u.) per min) of each fluorescence curve were calculated as the representation of their extracellular H 2 O 2 generation rate. At least three independent experiments were performed for statistical analysis.

ETs from disaggregated slug cells were quantified by the membrane-impermeant DNA dye PI (Cat: P4170, Sigma) in a microplate reader, at the final concentration of 0.5 μg ml−1. The intensity of PI-stained DNA was detected at 300 nm excitation and 590 nm emission. Similarly, the fluorescence intensity of Kp–GFP was detected at 400 nm excitation and 512 nm emission. DNase I was added to a final concentration of 100 U ml−1 for DNA digestion. Fluorescence signals of 13 different positions, covering 80% of the bottom area of each well were recorded and averaged every 5 to 20 min, depending on the sample size, for at least 4 h. At least three independent experiments were performed for statistical analysis.

Quantification of ETs from wild types and mutant strains was performed on disaggregated slug cells. About 5 μg ml−1 of K.p. or P.a. LPS was added to disaggregated AX4 and TirA-KO or AX2 and NoxABC-KO slug cells together with PI (0.5 μg ml−1) or SYTO9 (5 μM). The fluorescent DNA fibres (ETs) and cell numbers were counted at the indicated time points. The results were normalized as the number of ETs per 10,000 cells from more than 3 independent experiments.

Generation of KO strains

The NoxA, B and C triple KO strains were generated in the AX2 parent strain by the Cre-Lox technique (Supplementary Fig. 3). The construction of knockout vectors for NoxA, NoxB and NoxC shared the same strategy. The Blasticidin resistance (Bsr) cassettes from the original vectors described (generous gift of Dr B. Lardy (CNRS, France)23), were exchanged for the ‘LoxP-Bsr-LoxP’ cassette released from pLPBLP plasmid39. The pLox-NoxA vector was electroporated into AX2 cells, followed by PCR selection to obtain independent single NoxA-KO clones carrying the floxed Bsr cassette. To obtain double or triple KO clones using the same selection marker, the Bsr cassette was removed by transfecting the Cre recombinase expression plasmid pTX-NLS-Cre, leaving a LoxP site and three serial stop codons in both directions. This process was further repeated with the transfection of pLox-NoxB and then pLox-NoxC to obtain the NoxAB double KO and finally the NoxABC triple KO strains, respectively. The various KO clones were confirmed by genomic DNA PCR, using the primer pairs listed in Supplementary Table 1. Primers were designed to target the genomic DNA flanking each Nox gene sequence, so that the length of the PCR products can be used to confirm the successful clones due to the insertion and deletion of the Bsr cassette inside of each Nox gene.