In contrast, neurotensin (NTS) and urokinase plasminogen activator (uPA) display reduced EV uptake by MDA-MB-231 cells. Interestingly, EV uptake rate did not depend on the source of the EVs; breast cancer cells demonstrated no increase in uptake on administration of breast cancer-derived EVs in comparison to HEK293FT-derived EVs. Moreover, EV uptake was greatly enhanced by delivery in the presence of polybrene and spinoculation, suggesting that maximal EV uptake rates are much greater than those observed under basal conditions in cell culture. By investigating how the cell’s environment might provide cues that impact EV uptake, we also observed that culturing cells on soft matrices significantly enhanced EV uptake, compared to culturing on stiff tissue culture polystyrene. Each of these observations provides insights into the factors impacting EV uptake by breast cancer cells, while also providing a basis of comparison for systematically evaluating and perhaps enhancing EV uptake by various cell types. for 2?min at 4C and filtered through a 0.45-m-pore filter (VWR). Lentivirus was concentrated from filtered supernatant by ultracentrifugation at 100,420 for 90?min at 4C in a Beckman Coulter Optima L-80 XP ultracentrifuge with an SW 41 Ti rotor. Concentrated lentivirus was used to transduce 1.5??105 HEK293FT or MDA-MB-231 cells. Transduced cells were flow sorted on a BD FACSAria II flow cytometer to select for the top 50% of GFP-positive cells to create cell lines MH134 (HEK293FT CD63-CD-UPRT-EGFP) and MH135 (MDA-MB-231 CD63-CD-UPRT-EGFP). EV production, isolation, and characterization CDDO-EA EV-depleted medium was made by supplementing DMEM CDDO-EA with 10% exosome-depleted FBS (Gibco), 1% penicillin/streptomycin, and 4?mM l-glutamine. Alternatively, DMEM containing 20% FBS was cleared of EVs by ultracentrifugation at 120,416 for 135?min at 4C in a Beckman Coulter Optima L-80 XP ultracentrifuge with an SW 41 Ti rotor. The supernatant was then mixed with serum-free DMEM to achieve a final concentration of 10% FBS. To generate targeted EVs, HEK293FT cells were plated at 1??106 cells/mL in 15-cm dishes (18?mL of medium), and 6C8?h later, cells were transfected with 30?g CDDO-EA of targeting peptide-PDGFR transmembrane domain plasmid DNA and 1?g of DsRed-Express2 plasmid DNA (Clontech) as a transfection control using the CaCl2-HEPES-buffered saline method. Medium was changed to EV-depleted medium 16?h later. Transfection efficiencies were estimated by visualizing DsRed-Express2 fluorescence immediately before EV harvest. Typical efficiencies CDDO-EA were between 60% and 80%. For EV production, cells were cultured in EV-depleted Rabbit polyclonal to PROM1 medium for 24?h before conditioned medium harvest. EVs were harvested by differential centrifugation at 4C as previously described.17 Briefly, conditioned medium was centrifuged at 300 for 10?min (to remove cells), 2000 for 10?min (to remove cell debris and apoptotic bodies), and 10,000 for 30?min (to remove microvesicles). At each step, the supernatant was recovered for subsequent spins. EVs were pelleted from the final supernatant by ultracentrifugation at 120,416 for 135?min. EV concentration was determined by NTA using a NanoSight LM10-HS (Malvern) with a laser wavelength of 405?nm and software version 2.3. Samples were diluted in 1:50 or 1:100 in phosphate-buffered saline (PBS) to keep concentrations between 2 and 9??108 vesicles/mL. Samples were introduced manually. Three 30-s videos were acquired per sample at a camera level of 14 and processed at a detection threshold of 7. The blur, minimum track length, and minimum expected particle size were automatically set by the software. EV concentrations were defined as the mean of the concentrations determined from each video. Immunoblotting For western blot analysis, cells were lysed in RIPA buffer, and protein concentration was determined by BCA assay (Pierce). EVs were not lysed, and loads were normalized by vesicle count as determined by NanoSight. EVs and cell lysates were heated in Laemmli buffer at 70C for 10?min. Samples were run on 4C15% gradient polyacrylamide gel (Bio-Rad). Proteins were transferred to a PVDF membrane (Bio-Rad) at 100?V for 45?min. Membranes were blocked in 5% milk for 1?h at room temperature, blotted with the rabbit anti-FLAG antibody (ab1162; Abcam) diluted 1:1000, and incubated overnight at 4C. Primary antibodies were detected with the horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G secondary antibody (Thermo Fisher Scientific). Generation, characterization, and use of gelatin matrices An array of gelatin stiffnesses was generated by adding 200?L of gelatin dissolved in PBS to individual wells of a 48-well plate to produce surfaces.