Exo fbs 250a 1

The human microglia C20 cell line was donated by Dr. David Alvarez-Carbonell, Case Western Reserve University Cleveland, Ohio. Cells were cultured at 37 °C and 5% CO₂ in complete medium consisting of Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich, D6429), supplemented with 1 µM dexamethasone (PromoKine, PK-CA577-1042–1), 1% penicillin–streptomycin (Sigma-Aldrich, P4333), and 10% fetal bovine serum (FBS, Biowest, S181B). Hela cells were cultured at 37 °C, and 5% CO₂ in Minimum Essential Medium (ThermoFisher Scientific, 31095-029) complemented with 1% penicillin–streptomycin (ThermoFisher Scientific, 15140-122), 1% L-glutamate 200 mM (ThermoFisher Scientific, 25030-024), and 10% fetal bovine serum (FBS, Biowest, S181B). Upon EV stimulation, the complete cell culture media supplemented with 10% FBS (Biowest, S181B) was replaced by the EV-depleted media containing 10% EV-depleted serum (System Bioscience, EXO-FBS-250A-1) to avoid bovine derived EV contamination.

After BMSCs had grown to about 80% fusion, the culture medium was replaced by an exosome-depleted FBS-containing medium (EXO-FBS-250A-1; System Biosciences, Mountain View, CA, USA). The cells were then cultured a further 48 h. The medium was collected and centrifuged at 4 °C at 300×g for 10 min and at 2000×g for 10 min. After centrifugation, 0.22 μm Steritop™ (Millipore, Billerica, MA, USA) was used to remove the cells and cell debris. After that, the supernatant was transferred to the upper layer of an Amicon ultra-15 spinning Filter Unit (Millipore), and the supernatant liquid was centrifuged at 4000×g at 4 °C to about 200 μL. The supernatant was cleaned with PBS twice and then ultra-filtered to 200 μL. The liquid was placed on a 30% sucrose/D₂O buffer and placed in a sterile Ultra-Clear™ tube (Beckman Coulter, Brea, CA, USA) and centrifuged at 100,000×g for 60 min at 4 °C (Sorvall, Avanti J-26XP, fixed-angle rotor; Beckman Coulter). The fraction containing the BMSC-Exos was recovered using an 18-G needle, diluted in PBS, and centrifuged at 4000×g at 4 °C in centrifugal filter units until the final volume reached 200 μL. BMSC-Exos were stored at − 80 °C. The protein content of BMSC-Exos was measured by the bicinchoninic acid assay (BCA; Thermo Fisher, Waltham, MA, USA). A microplate reader (ELx800, BioTek, USA) was used to measure the absorbance at a wavelength of 562 nm.

Exosomes are traditionally isolated from conditioned media by serial centrifugation at a low speed, followed by ultracentrifugation at 100,000 × g to pellet the exosomes [73 , 74 (link)]. Recently, a proprietary method of exosome isolation was made commercially available and was reported to provide a rapid and efficient method for exosome isolation [23 (link), 75 (link), 76 (link)]. Briefly, cells were cultured for 48 hours; thereafter, media were replaced with RPMI 1640 supplemented with 10% exosome-depleted FBS [77 (link)]. (EXO-FBS-250 A-1, System Biosciences). After 24 hours, cells and debris were removed by centrifugation at 350 g for 10 minutes and 2000 g for 30 minutes. This was followed by filtration of the supernatant with 0.2 μm filters. Next, each sample was combined with ½^th volume of total exosome isolation reagent (Invitrogen, Life Technologies). Cell medium and the exosome isolation reagent were mixed by brief vortexing until a homogeneous solution was formed and incubated at 4°C overnight before centrifugation at 4°C at 10,000 g for 1 hour. The supernatant was aspirated and discarded, and the exosome pellet was resuspended in phosphate-buffered saline (PBS) and stored at −20°C until further use.

M0 and M1 macrophages were cultured in RPMI 1640 medium containing exosome-depleted FBS (#EXO-FBS-250A-1, System Biosciences, CA, USA). Exosomes were precipitated from the cultured medium using the Total Exosome Isolation Reagent (#4478359, Invitrogen, CA, USA). Briefly, the medium was centrifuged at 2500 rpm for 30 min to remove cells and debris. The supernatant was collected and passed through a 0.22-μm-pore filter (#SLGVR33RS, Merck Millipore, MA, USA). Subsequently, the medium was mixed with the Total Exosome Isolation Reagent and incubated overnight at 4 °C. Exosomes were finally pelleted by centrifugation at 13,000 rpm for 1 h at 4 °C, re-suspended in PBS, and stored at −80 °C. The exosome protein content was quantified using a Pierce™ BCA protein assay kit (#23227, Thermo Fisher Scientific, Waltham, USA). The expression levels of exosomal marker proteins CD63, CD81, and heat shock protein 70 (Hsp70) were assessed using western blotting, as described in the Western blot analysis section.
The size distribution and concentration of purified exosomes were measured using nanoparticle tracking analysis with a Malvern NanoSight NS300 (Malvern Instruments Company, Malvern, England) and the corresponding software NanoSight NTA 3.4 Analytical.

PC3 and MG63 cells were procured grown in normal media at at 37 °C and 5% CO2. At 50% confluency in 150 mm culture dishes, cells were washed thrice with PBS then supplemented with media containing 5% exosomes depleted FBS (System Biosciences Cat# EXO-FBS-250A-1). After 48 h, culture supernatant was collected and centrifuged twice at 1000×g for 10 min to remove cellular debris. 15 ml of cleared media was then concentrated to ~ 1 ml by centrifugation at 14,000×g for 30 min using Amicon^® Ultra-15 centrifugal filter unit (Sigma Cat# UFC901008). Concentrated media was then was overlaid on 35 nm qEV-original size exclusion columns (Izon Cat# SP5) followed by elution with PBS. 500 μl fractions were collected using an automatic fraction collector from Izon Science and Protein concentration was determined by NanoDrop A280. Only fractions that contained low protein concentrations were used for subsequent experiments. Purity, size, and concentration of all preparations of exosome fractions were analyzed using nano-tracking analysis (NTA) on a NanoSight LM10 (Malvern Panalytical). Particle counts and size was done with the NTA 3.1 software using a 50 μl/min flow rate and read at 25 °C on a 488 nM laser/filter. All NTA readings were performed using PBS which had been previously depleted of nanoparticle background by filtration through a 0.02 µM membrane.