0.45 μm strainer

BMDM were differentiated from bone marrow incubated in DMEM with 10% fetal calf serum (FCS), 2 mM L-glutamine, 10U/ml Penicillin, 10 μg/ml Streptomycin, and 10 ng/ml M-CSF (GenScript) for 7 days.
NIH 3T3 cells (mouse, male, ATCC CRL-1658) were maintained in DMEM supplemented with 10% Newborn Calf Serum (NBCS), 2 mM L-glutamine, 10U/ml Penicillin, and 10 μg/ml Streptomycin.
Primary corneal epithelial cells were collected from C57Bl/6 mice by incubating eyes overnight at 4 °C in a 1:1 mixture of DMEM:F12 supplemented with 4 mg/ml Dispase I (Sigma–Aldrich), 10U/ml Penicillin, 10 μg/ml Streptomycin, and 0.025 μg/ml Amphotericin B. Epithelial sheets were peeled from eyes and dissociated in TrypLE for 10 min at 37 °C with gentle agitation. Cells were passed through a 0.45 μm strainer (Corning), washed, and cultured on collagen and fibronectin-coated plates in KSFM supplemented with 5 ng/ml hEGF, 50 μg/ml Bovine Pituitary Extract, and 100 ng/ml Cholera Toxin.
All cells were maintained at 37 °C with 5% CO₂. Adherent cells were detached for subculture and experiments using TrypLE. Unless otherwise noted, all cell culture materials were purchased from Gibco/Thermo Fisher.

EVs isolation was performed according to a previously described protocol [97 ]. In brief, USCs were washed twice with PBS in a T75 flask and cultured in serum–free expansion media. The conditioned media was collected after 48 h and run through successive centrifugation steps with increasing centrifugation forces and durations aiming to isolate EVs, based on their sizes as follows: centrifuge at 300× g for 10 min to precipitate cells, followed by the first centrifuge for the supernatant at 2000× g for 20 min at 4 °C to precipitate any dead cells. The supernatant was collected and centrifuged at 10,000× g for 40 min at 4 °C to precipitate cell debris. Furthermore, the supernatant was filtered through a 0.45 μm strainer (Corning, NY, USA). Finally, the EVs were precipitated by ultra–centrifugation (Beckman Coulter Type,70 Ti rotor, Brea, CA, USA) at 100,000× g for 90 min at 4 °C [98 (link)]. The formed pellet was washed once with PBS using the same speed to eliminate concomitant proteins from the EV pellet. The obtained EV pellet was resuspended in 100 µL PBS.
The absence of Mycoplasma and Acholeplasma contamination to the USC and the isolated vesicles was confirmed by PCR analysis of the supernatant following a previously described protocol [99 ].