A merged image (panel 3) showing few areas of co-localized ICAM-1 and CD11c staining (yellow) and little relocalization of CD11c from your cell surface to the cytosol. PDAC cell lines differed in their surface proteins, lipid composition, and efficiency of fusing with THP-1-derived macrophages model commonly used to examine TAM-tumor cells interactions [15]. AsPC-1 cells were cultured in altered RPMI 1640 made up of 10% FBS, 10 mM HEPES, 1 mM sodium pyruvate, and 13.9 mM D-glucose. Both THP-1 and BxPC-3 cells were cultured in 10% FBS-containing RMPI 1640, and PANC-1 and MIA-PaCa2 cell lines were cultured in 10% FBS-containing DMEM. The HPDE cell collection H6c7, a gift from Dr. M.S. Tsao, University or college Health Network in Toronto, was managed in keratinocyte serum-free medium (ThermoFisher Scientific) ME-143 [16]. Each cell collection was seeded into a 10-chamber CellSTACK manufacturing plant (Corning Inc.), and at 80% confluence standard culture medium was replaced with serum-free medium. After 48 hours, spent cell culture medium (SCM) was collected and utilized for subsequent exosome purifications. ME-143 Exosome isolation To eliminate cellular debris that could contaminate downstream analysis of exosomal proteins, lipids, or secreted factors, sequential centrifugation was used to purify the secreted exosomes. SCM was centrifuged twice at 500 x for 10 minutes at 4C to pellet large cellular debris, and smaller debris was then pelleted at 10,000 x for 30 minutes. The final supernatant was loaded into thinwall polypropylene ultracentrifuge tubes (10 mL/tube) (Beckman Coulter Inc.), underlayed with 20 mM Tris/30% sucrose in deuterium oxide (1 mL/tube), and centrifuged at 100,000 x for 90 minutes at 4C to pellet the exosomes. The tubes were pierced through the bottom with an 18-gauge needle and the sucrose layer was drawn into the syringe. The sucrose layers were pooled and diluted with excess 1X calcium- and magnesium-free phosphate buffered saline (PBS), and the exosomes were again pelleted at 100,000 x for 90 minutes. The exosome pellet was resuspended in PBS and stored at -80C. Exosome protein concentration was determined using a NanoOrange Protein Quantitation Kit (ThermoFisher Scientific), and total exosomal protein was used to normalize all other exosome comparisons. Exosome size analysis and visualization of exosomes by transmission electron microscopy (TEM) Exosome size was measured using a Zetasizer Nano S (Malvern Instruments Ltd.). For TEM, 5 L of exosome suspension was placed on a piece of parafilm and a formvar-coated copper grid was floated on the drop for 20 minutes at room temperature. The copper grid was blotted quickly on filter paper, placed on 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.3, and washed by transferring to three separate PBS drops for one minute each. After placing in 1% glutaraldehyde in 0.1 M sodium phosphate buffer for 5 minutes, the grid was blotted quickly and moved to distilled water for 2 minutes. The grid was then washed four times with PBS and placed in 1% uranyl acetate for 20 seconds. Excess uranyl acetate was removed by blotting and the grid was imaged by transmission electron microscopy on a JEM-1400Plus (JEOL USA, Inc.). Immunoblot analysis of exosomal proteins Equivalent amounts of total exosomal protein (30 g) were resolved by SDS-PAGE and transferred to a polyvinylidine fluoride membrane. Primary antibodies used were: ICAM-1 (Cell Signaling Technology, #4915), flotillin-1 (D2V7J, Cell Signaling Technology, #18634), EpCAM (D1B3, Cell Signaling Technology, #2626), and CD9 (D8O1A, Cell Signaling Technology, #13174). Primary antibodies were diluted 1:1,000 in 5% BSA/TBST, and secondary HRP-conjugated antibodies were diluted 1:5,000 in 5% BSA/TBST. Target proteins were detected with an enhanced chemiluminescent substrate (ThermoFisher Scientific). The pan-exosomal marker flotillin-1 was Rabbit Polyclonal to CDK5RAP2 used as a loading control. STtimulated emission depletion (STED) microscopy THP-1 monocytes were differentiated into non-polarized (M0) macrophages with PMA (Cayman Chemical) [17]. After treating with 150 nM PMA-containing growth medium for 24 hours, PMA-containing medium was replaced with standard culture media and the THP-1 cells were allowed to recover for 24 hours. For co-localization studies, PMA-differentiated THP-1-derived macrophages were treated with 30 g of AsPC-1 exosomes. After 4 minutes, cells were rinsed three times with PBS and fixed with ice-cold 100% methanol for 5 minutes. Following fixation, cells were washed three times with PBS for 5 minutes, blocked with 2% BSA in PBS and incubated with primary antibodies against CD11c (Invitrogen, #MA11C5, host: hamster) and ICAM-1 (Cell Signaling Technology, #4915T, host: rabbit) diluted 1:250 in 2% BSA/PBS at 4C overnight. Cells were washed three times with PBS for 5 minutes ME-143 and incubated for 1 hour with Alexa Fluor 568-conjugated goat anti-hamster secondary antibody (Invitrogen, #A-11011) and Alexa Fluor 532-conjugated goat anti-rabbit secondary antibody (Invitrogen, #A-11009) each diluted 1:250 in 2% BSA/PBS. After washing with PBS for 5 minutes, coverslips were mounted using ProLong Gold Antifade (Invitrogen, #”type”:”entrez-protein”,”attrs”:”text”:”P36930″,”term_id”:”1248281091″,”term_text”:”P36930″P36930). Slides were kept at 4C protected from light prior to imaging. STED sub-diffraction microscopy was performed using Leica AOBS SP8 ME-143 system integrated with 3XSTED module. STED images of fluorescently labeled cells were acquired using a STED 100x/ 1.40.