Amicon ultra 15 100 kda filter

Reelin was obtained as conditioned media from a cell line (HEK293T) that stably expresses mammalian Reelin [104 (link)]. Cells were cultured in DMEM high glucose (Gibco) supplemented with fetal bovine serum (10% FBS, Biological Industries), 1% penicillin/streptomycin (Gibco), and G418 (0.5 mg/ml). In order to collect Reelin, the culture media is serum-free. The supernatant containing Reelin was collected for 5 days, and the medium was concentrated using Amicon ultra-15 100-kDa filters (Millipore). Reelin concentration in the medium was estimated semiquantitatively: BSA dilution curves and an aliquot of Reelin-containing medium were run through an SDS-PAGE. The gel was stained with Coomassie blue to visualize reelin bands. After that, the bands corresponding to BSA and Reelin were digitalized to analyze intensities in Fiji [105 (link)].

The HeLa and HT1080 cell lines were purchased from Sigma-Aldrich (ECACC, 93021013 and 85111505). Cells were cultured until 80% confluency and washed 3 times with DMEM. Then, the medium was switched to DMEM. They were then conditioned for 48 hours, after which exosomes were isolated as previously described11,12. In brief, exosomes were isolated by ultrafiltration. Conditioned medium was filtered through 0.22 µm Steriflip filters to remove cellular debris and large vesicles. The filtrate was then added to Amicon Ultra-15 100 kDa filters (Millipore, SCGP00525) to centrifuge at 5,000 × g for 5 min. The flow-through was discarded and the concentrated exosomes were collected and washed with PBS three times before being stored at -80°C.
Exosome labeling was performed using 10 uM DiI or DiO (Thermo Fisher, V22889), incubated for 20 minutes at 4°C. Then, Exosome Spin Columns (Thermo Fisher, 4484449) were used to remove the unincorporated dye.

Cardiac stem cells (CSCs) were generated and cultured using the cardiosphere method as previously described [2 (link), 15 (link)]. In brief, passage 4 hCSCs were cultured in IMDM containing 20% fetal bovine serum (FBS) until 90% confluency; then medium was switched to serum-free Iscove's Modified Dulbecco's Medium (IMDM). Medium was conditioned by the CSCs for 15 days, then removed, and stored at −80°C awaiting further processing.
For exosome isolation, CSC-conditioned medium was thawed slowly on ice overnight; then exosomes were isolated using ultrafiltration [19 ]. Briefly, 10 mL exosome conditioned medium was filtered through 0.22 μm vacuum sterilization filters (Millipore, Billerica, MA) to remove larger vesicles and cellular debris. The filtrate was then added to Amicon Ultra-15 100 kDa filters (Millipore, Billerica MA) and spun at 4000 ×g for 10 min. Labeling was performed using 10 μM DiI (Life Technologies, Carlsbad, CA) for 30 min. Filtrate was discarded, and volume of retentate was adjusted to 15 mL with PBS. Spinning and washing with PBS was repeated two more times. On the last cycle, remaining volume containing the exosomes (approximately 1 mL) was transferred to micro centrifuge tube and stored at −20°C.