, MHC class II [1B5 hybridoma anti-DRa (39)], actin (clone c4, Millipore), ADAM10 (clone #163003, R&D Systems), HSP70 (clone C92F3A-5, Enzo Life Science), MHC class I [HC10 (40) + HCA2 (41) hybridomas], anti-TSG101 (clone 51/TSG101, BD Biosciences); polyclonal mouse anti-human ALIX (Abnova); rat monoclonal anti-human HSC70 (clone 1B5, Enzo Life Sciences), and GP96 (clone 9G10, Enzo Life Sciences); rabbit polyclonal anti-human Annexin II (Genetex), Reagents. Antibodies for Western blot were: mouse monoclonal anti-human CD63 (clone H5C6, BD Biosciences), CD9 (clone CBL162, Millipore), CD81 (clone B-11, p.4
, Ultrapure Sucrose and iodixanol (Optiprep) were from Sigma. Lipid staining was performed with FM 4-64 FX dye (Life Technology): briefly cells were seeded on poly-L-lysine-coated glass coverslips overnight, placed on ice
, On day 5 of culture (DCs) or when subconfluence was reached (cell lines), cells were washed in PBS (Gibco) and further cultured in EV-depleted medium (10% EV-depleted FCS final) or in DMEM-Glutamax without FCS (MDA-MB-231 exposed to starvation) for 24 h before collection of conditioned medium for EV isolation. EVs were isolated by differential ultracentrifugation as previously described (11) (Fig. 1A). Briefly, conditioned medium was centrifuged at 300 × g for 10 min at 4°C to pellet cells. Supernatant was centrifuged at 2,000 × g for 20 min at 4°C (2K pellet), transferred to new tubes, and centrifuged in a 45Ti rotor (Beckman) for 40 min at 10,000 × g (9,000 rpm = 10K pellet), and finally for 90 min at 100,000 × g (30,000 rpm = 100K pellet). All pellets were washed in 50-60 mL of PBS and recentrifuged at the same speed before being resuspended in 50-100 ?L of sterile PBS. Cells recovered from the first 300 × g pellet were pooled with cells detached from the plates by incubation at 4°C in PBS-EDTA (DCs) or in trypsin-EDTA (adherent cells, EV Isolation. Bovine EV-depleted medium was obtained by overnight ultracentrifugation at 100,000 × g in a 45Ti rotor, of medium supplemented with 20% FCS
transferred to a SW55Ti rotor tube (Beckman), and mixed 1:1 with 60% (wt/vol) stock solution of iodixanol/ Optiprep. A 40% iodixanol working solution was prepared [40% (wt/vol) iodixanol, 0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA, final pH set to 7.4] and used to prepare 20% and 10% (wt/vol) iodixanol solutions. Next, 1.3 mL 20% (wt/vol) iodixanol and 1.2 mL 10% iodixanol were successively layered on top of the vesicle suspension and tubes were centrifuged for 1 h at 4°C at 350,000 × g (54,000 rpm) in SW55Ti (stopping without break); 10 fractions of 490 ?L were collected from the top of the tube. Density was assessed with a refractometer (Carl Zeiss). Fractions were diluted with 2.5 mL PBS and centrifuged for 30 min at 100,000 × g (43,000 rpm) in a TLA 110 rotor, 1.5 mL buffer containing: 0.25 M sucrose, 10 mM Tris pH 8.0, 1 mM EDTA (pH 7.4) ,
, M sucrose solution in PBS were used to prepare 15 fractions with sucrose molarity ranging from 0.4 to 2.0 M. Pellets mixed with 2.5 M sucrose to 1.5 mL total volume were loaded at the bottom of a SW40Ti tube and the gradient was layered by pouring sequentially 750 ?L of each of the 15 solutions, Sucrose gradients were built manually as described in ref. 42. Briefly 2.0 M and 0.4
, Beads were then washed three times with 500 ?L of PBS-0,001% Tween, resuspended in 500 ?L of the same buffer, to which sEVs from 15 million DCs in 25 ?L PBS were added, followed by overnight incubation at 4°C with rotation. Bead-bound EVs were collected and washed three times in 500 ?L PBS-Tween. Nonbead-bound supernatant was pooled with the supernatants of bead washes and centrifuged for 30 min at 100,000 × g (43,000 rpm) in a TLA 110 rotor (Beckman, Optima TL100 centrifuge), to generate concentrated FT. FT and bead-bound EV samples were resuspended in 30 ?L of loading buffer 1× (Invitrogen) and boiled at 95°C for 5 min before loading on gel, Antibodies for immuno-isolation [mouse monoclonal antihuman CD63 (clone H5C6, BD Biosciences)
, Protein concentration was measured using Micro-BCA (Thermo Scientific) for both cell lysates and vesicle preparation in the presence of 0.2% SDS. Equivalent micrograms of proteins (Figs. 1 and 4 B and C and Fig. S1D), or pellets recovered from a given number of cells [i.e., a given volume of conditioned medium) (Figs. 4D and 5B and Fig. S3 B and D) were loaded for all samples from, Triton X-100, 0.1% sodium azide with a mixture of antiproteases
, × 10 6 (Fig. 5B), or 10-25 × 10 6 cells (Fig. 4D and Fig. S3 B and D) was analyzed. Separation was performed under nonreducing, Materials
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