The human mast cell line (HMC-1) was cultured in IMDM complemented with 10% EV-depleted fetal bovine serum (FBS), 100 units/ml streptomycin, 100 units/ml penicillin, 2 mM L-glutamine and 1.2 U/ml alpha-thioglycerol (all regents were from Sigma, St Louis, MO, USA). FBS was depleted of EVs by ultracentrifugation at 118,000 × gavg for 18 h at 4°C (Type 45 Ti rotor, 38,800 rpm, k-factor 178.6, Beckman Coulter, Brea, CA, USA).
Mouse lung tissue
Lungs from one mouse (C57BL/6) were dissected and immediately immersed into a petri dish filled with RPMI (Sigma Aldrich) supplemented with 100 units/ml Penicillin, 100 µg/ml Streptomycin and 2 mM L-glutamine (all regents were from HyClone GE healthcare Life Sciences, Logan, Utah, USA). The lungs were sliced into small cubes (1–2 mm) and incubated with Collagenase D (Roche Stockholm, Sweden) (2 mg/ml) and DNase I (Roche, city, country) (40 U/ml) dissolved in RPMI plain medium (Sigma Aldrich) for 30 min at 37°C. Chopped lung pieces were filtered (70 µm pore size) and the flow through put in culture for 72 h at 37°C in RPMI (Sigma Aldrich) supplemented with 100 units/ml Penicillin, 100 µg/ml Streptomycin, 2 mM L-glutamine (all regents were from HyClone), 20 µg/ml Carbenicillin and 10% FBS (Gibco Invitrogen Corporation, Carlsbad, CA, USA). FBS was depleted of EVs prior to use as described earlier.
20 ml of whole blood was collected from a healthy volunteer into BD vacutainer SST tubes (BD biosciences, New Jersey, USA) and left to clot for 1 h at room temperature to be able to collect serum. The blood was then centrifuged at 1880 × g for 10 min. The serum was transferred to ultracentrifuge tubes and further handled as described below.
Cells of strain BY4741 were grown to OD600 0.5 in synthetic complete medium. Vesicles were isolated from 900 ml of culture using a centrifugation-based protocol. Cells were removed by pelleting them first at 400 × g for 10 min and at 600 × g for 15 min. The supernatant was used for further vesicle isolation.
All EVs were isolated by differential ultracentrifugation. Cells were pelleted at 300 × g for 10 min. Supernatant was transferred to ultracentrifuge tubes and centrifuged at 16,500 × gavg for 20 min to pellet what is usually referred to as microvesicles. The supernatant was again carried over to new ultracentrifuge tubes and centrifuged at 118,000 × gavg for 3.5 h at 4°C to pellet what is usually referred to as exosomes. Both microvesicle and exosome pellets were resuspended in phosphate buffered saline (PBS) and used immediately for downstream experiments. For EM experiments. Exosomes were further purified by floatation on a density gradient.
Cryo-EM images were acquired for vesicles derived from ejaculate and HMC-1 cultures, with either of two electron microscopes. One was a Tecnai F30 electron microscope (FEI Company Ltd.) with a GATAN UltraCam detector, lens coupled, 4 K CCD camera attached to a Tridiem Gatan Image Filter (Gatan, Inc., Pleasanton, CA, USA). The images were acquired at 27,500 X magnification with -4 to -6 defocus. The other electron microscope used was a Philips CM120 BioTWIN.
Thin section electron microscopy: 16,500 × g pellet were resuspended in 20% bovine serum albumin (BSA) in PBS and high pressure frozen (Leica Empact I, Leica Microsystems) directly after isolation. Samples were freeze substituted at -90°C for 1 h (solution: 2% uranyl actetate (UA) in 10% methanol and 90% acetone), then washed twice with acetone. Temperature was increased 3°C/h to -50°C where the infiltration of HM20 was performed before 48 h ultra violet light polymerization. 70 nm thick sections were cut and en-section stained with uranyl acetate and lead citrate before being imaged on a Leo 912AB Omega TEM operated at 120 kV.
EV isolates from either HMC-1, blood, yeast or lung tissue were loaded onto glass bottom dishes and left to adhere to the surface for 15 min at room temperature. The dishes were gently washed 3 times with PBS. They were then stained with PKH67 by mixing of the lipid dye with the Diluent C in a 1:500 ratio, of which 150 µl was placed on each dish containing vesicles. The dye was left to incubate on the dishes for 2 min after which it was removed and then the dishes were washed 3 times with PBS and finally filled with 2 ml of fresh PBS. The samples were then immediately analyzed with an Axio Observer (Zeiss, Oberkochen, Germany) with which time lapse series were taken at a magnification of 63X.
Samples were incubated over night under gentle agitation with dynabeads coated with antibodies against CD63 (15 µg exosomes/70,000 beads/antibody; Human CD63 Isolation/Detection (from cell culture media), Thermo Fisher Scientific). The sample was washed with PBS containing 1% EV depleted FBS after which the samples were incubated with human IgG (Sigma-Aldrich) for 15 min at 4°C. The samples were again washed and then incubated with PE-labeled antibodies against CD9, CD63, CD81 as well as with a PE(phycoerythrin)-labeled isotype control (BD Bioscience, Erembodegem, Belgium) for 40 min with gentle agitation. Samples were again washed before analysis with a FACSVerse (BD Biosciences).