0.2 m filter

Exosomes were isolated by a differential ultracentrifugation method as previously described with some modifications^{27 (link)}. Briefly, the BALF from three sex- and treatment-matched mice were pooled and centrifuged at 500g for 5 min to sediment the BALF immune cells. The cell-free supernatant was further centrifuged at 3000g for 10 min to sediment the dead cells. Then, the supernatant was centrifuged at 10,000g for 70 min using SW28 rotor (Beckman Coulter Optima L-90 K Ultracentrifuge). The pellet comprising of cell debris and large microvesicles was discarded and the supernatant was filtered using a 0.2 µm filter (VWR, Radnor, PA). The filtered supernatant was further centrifuged at 100,000g for 100 min. The supernatant was carefully discarded without disturbing the exosome pellet. The pellet was resuspended in 200 µl of phosphate-buffered saline (PBS). Nanoparticle tracking analyses (NTA; Nanosight 300) on vesicular population harvested using differential ultracentrifugation method typically have a diameter of 131.2 ± 4.4 nm (mean ± SEM) and 102.4 ± 5.8 nm (mode ± SEM). Resuspended exosomes were snap-frozen and stored at − 80 °C. All the centrifugation steps were performed at 4 °C.

Overnight cultures of lactobacilli were grown without agitation in MRS medium at 37 °C. Cell-free supernatant was prepared by centrifuging the culture at 2000 × g for 10 min at 4 °C and then filtering through 0.2 µm filters (VWR, Haasrode, Belgium).

All of the centrifugations and ultracentrifugations were performed at 4°C. Tracheal supernatants from EV samples were centrifuged at 300× g for 10 min. to remove cellular debris. Subsequently, supernatants were recovered and centrifuged at 2000× g for 20 min. Supernatants were again recovered and ultracentrifuged at 10,000× g for 30 min. (Optima L-100XP, Beckman Coulter, Mississauga, ON, Canada). Supernatants were recovered and filtered with 0.2 µm filters (VWR, Montreal, QC, Canada). Supernatants were then ultracentrifuged at 100,000× g for 60 min. (Optima L-100XP, Beckman Coulter, Mississauga, ON, Canada). Supernatants were discarded and pellets were resuspended in FBS-free complete Medium 199 and ultracentrifuged at 100,000× g for a final 60 min. (Optima L-100XP, Beckman Coulter, Mississauga, ON, Canada). The supernatants were discarded, and the pellets were resuspended in 200 µL of phosphate-buffered saline (PBS, Gibco, Burlington, ON, Canada). Finally, for miRNA isolation from EV samples, 700 µL of QIAzol reagent (QIAGEN, Toronto, ON, Canada) were added to samples that were designated for RNA isolation and then stored at −80 °C. Samples designated for Western Blot and transmission electron microscopy analyses were stored at −80 °C in PBS.