Dnase 1

Testis cells were prepared for FACS analysis based on previous studies with minor modifications^{38 ,39 (link)}. Briefly, decapsulated testes were incubated in DMEM medium supplemented with 1 mg/mL Collagenase type IV (Sigma) and 10 μg/mL DNase I (Roche) at 33 °C for 20 min. Then 1 mg/mL trypsin (Gibco) was added and samples were incubated for another 20 min, with the tube being inverted several times. The dissociated testis samples were pipetted with a plastic disposable Pasteur pipet several times, followed by adding 10% FBS to inactivate trypsin. For PI staining of fixed cells, testis cell samples were centrifuged and pellets were resuspended in 100 μL PBS and then 900 μL 70% ethanol was added. Cells were fixed overnight at 4 °C, washed once with PBS, resuspended in PI staining solution (PBS supplemented with 0.1% TritonX-100, 200 μg/mL DNase-free RNase and 20 μg/mL PI), incubated at 37 °C for 30 min, and then used for FACS analysis with a BD FACS Calibur Flow Cytometer. For Hoechst 33342 staining, dissociated testis cell samples were incubated with 100 μg/mL Hoechst 33342 and 10 μg/mL DNase I at 33 °C for 1 h and then passed through 40-μm cell strainers (BD Falcon). Cell samples were added with 10 μg/mL PI and immediately subjected to FACS analysis with a BD Influx cell sorter. Examples of the FACS gating strategy are shown in Supplementary Figure 8a, b.

One week after the last vaccine dose, we euthanized mice and removed the spleen and lungs to assess immune responses. Single-cell suspensions of splenocytes were prepared by gently pressing the cells out of the spleen sac; lysing red blood cells with PharmLyse (BD Pharmingen); washing the cells; and filtering through a 70 µm nylon cell strainer (Falcon). Single-cell suspensions of lung cells were prepared by cutting the lung into small pieces with a scalpel; incubating at 37°C for 1 h with shaking in 10 mL of digestion solution (300 U/mL collagenase type II [Worthington] and 0.15 mg/mL DNase I [Worthington] in PBS); filtering through a 40-µm nylon cell strainer (Falcon); lysing red blood cells with PharmLyse (BD Pharmingen); and washing the cells. Advanced RPMI-1640 (Invitrogen) supplemented with 2% heat-inactivated fetal bovine serum, 2 mM glutamine dipeptide (glutaGRO Supplement, Corning), 10 mM HEPES buffer, 50 µM β-mercaptoethanol, and penicillin (100 IU/mL)-streptomycin (100 µg/mL) was used as the medium.

For DNase treatment, P. putida was grown as described above (microcosm on AA7075-T6 incubated for
4 weeks). After 4 weeks incubation coupons with attached microbial
communities were incubated in humid air for 15–30 min to allow
the fuel vapor to evaporate. DNase I at a concentration of 270 U/mL
(ITW reagents) was added into a 10 mL Falcon tube containing DNase
I buffer with the coupon and then incubated at 37 °C for 48 h.
For controls, distilled water was added instead of DNase I. After
incubation with DNase I, the liquid phase was removed, coupons were
washed four times with 0.9% (w/v) NaCl, and the cells were stained
with SYTO-9.