Bca protein kit

Differential ultracentrifugation was performed to isolate and purify EVs, as previously described [14 ]. Briefly, the supernatants from M2 microglia culture were first centrifuged at 300 g for 10 min, followed by centrifugation at 2000 g for 10 min to remove cells and dead cells. Subsequently, the remaining supernatants were centrifuged at 10,000 g for 30 min to remove debris. The final supernatants were ultracentrifuged at 100,000 g for 70 min to obtain the EVs. To eliminate contaminating proteins, the EVs were washed once with PBS and subjected to ultracentrifugation at 100,000 g for 70 min. Finally, the EVs were resuspended in PBS and stored at -80°C. The protein concentration of the EVs was determined using a BCA protein kit (#ZJ102, Epizyme). The particle diameter and concentration of EVs were assessed using Nanoparticle Tracking Analysis and Transmission Electron Microscopy. To label the EVs, the PKH26 Red Fluorescent Cell Linker Kit (#PKH26GL-1KT, Sigma-Aldrich) was utilized with minor modifications [15 (link)]. In brief, EVs and 2 μl of PKH26 dye were added to 500 μl of Diluent C, and the mixture was incubated for 4 min at room temperature in the dark. To eliminate excess dye, 200 μl of FBS was added, and the labeled EVs were washed with PBS at 100,000 g for 70 min. The EVs pellet was resuspended in PBS for further in vitro and in vivo assays.

Brain tissues in the peri-ischemic area and primary astrocyte were used for western blot analysis. The protein concentration was measured using a BCA protein kit (Epizyme). The proteins (30 μg/group) were separated into a 5-10% gradient SDS-PAGE gel and then transferred onto PVDF membranes. After blocking with 5% milk, the bands were incubated overnight at 4°Cwith primary antibodies, including anti-CD63 (1:1000, #sc-15363, Santa Cruz Biotechnology), anti-TSG101 (1:1000, #ab83, Abcam), anti-TNF (1:500, #sc-52746, Santa Cruz Biotechnology), anti-MMP3 (1:1000, # 17873-1-AP, ProteinTech Group), anti-NFκB p65 (1:1000, #8242T, Cell Signaling Technology), or anti-β-actin (1:1000, #66009, ProteinTech Group). Then the membranes were incubated with HRP-conjugated secondary antibody (1:5000, #HA1006 or #HA1001, HUABIO) at room temperature for 1 h. Visualization analysis was performed using an enhanced chemiluminescence substrate (#SQ201, Epizyme) and an imaging system (Bio-Rad, Hercules, CA).