Bca protein assay kit

Before EV isolation, hBM-MSCs at about 85% confluent were thoroughly washed with complete culture medium and then cultured under 20% O₂ (normoxic) OR 1% O₂ (hypoxic) conditions for 24 h, followed by EV isolation as described by Lo Sicco et al. [15 (link)] with minor modifications. Briefly the cell culture media was centrifuged at 300×g for 5 min at room temperature, 2000×g for 30 min at 4 °C and 10,000×g for 30 min at 4 °C to remove cells and cell debris. The supernatant was centrifuged at 100,000×g for 60 min at 4 °C, and the pellet was resuspended in sterile PBS, followed by another centrifugation at 100,000×g for 60 min at 4 °C to isolate MSC-EV. MSC-EV were re-suspended in sterile PBS before use. MSC-EV aliquots were equilibrated for EV density based on their total protein concentration measured using a BCA protein assay kit (GeneCopoeia) following manufacturer’s instructions. EV precipitation from cell culture media was confirmed by western blot detecting EV marker CD9 and CD81 (Additional file 1: Figure S1).

Total protein concentrations were measured using a BCA Protein Assay Kit (Genecopoeia, USA). Frozen brain samples were mechanically homogenized and centrifuged at 12,000 rpm for 15 min at 4 °C. The levels of interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), and interleukin-1β(IL-1β) were measured using specific ELISA kits (eBIOSCIENCE, USA) according to manufacturer instructions. The concentration of the cytokines was determined by color intensity measured by spectrometry in a micro ELISA reader (Varioskan Lux, Thermo Scientific). The results are expressed as picogram per milligram for tissue samples.