Ca2 and mg2 free pbs

Splenocytes, lymph nodes, and thymi were prepared as single cell suspensions using 70 μm cell strainers in RPMI 1640 (HyClone) supplemented with 10% fetal bovine serum (FBS) (HyClone), penicillin (100 IU)-streptomycin (100 μg mL^-1) (Sigma), β-mercaptoethanol (0.05 mM) (Gibco) (complete RPMI). Splenocytes and thymocytes were washed once in Ca²⁺- and Mg²⁺-free PBS (Sigma), treated with 1 ml of red blood cell lysis buffer for 1 minute, and washed twice in PBS before further processing. Peritoneal cells were obtained by injecting and withdrawing 8-10 ml of PBS into the peritoneal cavity. To obtain lymphocytes from blood for flow cytometry, 2-3 drops of blood collected in 1.5 mL Eppendorf tubes containing 20 µL EDTA. Blood was treated twice with 2 ml red blood cell lysis buffer for 4 to 5 minutes and washed in PBS before further processing. For ELISA, blood was allowed to coagulate at room temperature for one hour, spun down at 6000 rpm for 6 minutes, and serum was collected and stored at -20°C.

For the evaluation of HGF secretion by hUCMSCs in vitro, cells at passage 3 were received pre-treatment with edaravone or DEM as described above and then washed thrice with Ca2⁺ and Mg2⁺-free PBS (Sigma-Aldrich), and cultured in 10 ml DMEM supplemented with 0.05% bovine serum albumin (BSA, Sigma-Aldrich) for 24 hours. After that, collected and concentrated conditioned medium was subjected to HGF ELISA measurement (RayBioteck, Norcross, GA). For in vivo measurements, paraffin embedded liver tissues at day 7 post-challenge were prepared for immunohistochemical staining of HGF. HGF-secreting cells were labeled with HRP/DAB system (Zhongqiao, Beijing, China). Hematoxylin was used as the counterstain of cellular nuclei.