Pbs phosphate buffered saline pbs

The study was approved by the local ethics committee. hFM-MSCs were isolated from 5 placentas obtained from 5 healthy donors by caesarean sections, after written informed consent in accordance with the Declaration of Helsinki, as previously described (Chatgilialoglu et al., 2017 (link)). Briefly, the amniochorionic membrane was separated from the chorion plate and washed in PBS phosphate-buffered saline (PBS, Corning, Somerville, MA, USA) supplemented with 1% of penicillin-streptomycin (Thermo Fisher Scientific, Walthman, MS, USA). The membrane was fragmented into small pieces and digested using a solution of 1 mg/mL collagenase type IV (Sigma-Aldrich, St. Louis, MO, USA) and 0.25% trypsin-EDTA (Corning) for 30 min at 37°C in agitation. After incubation, the digestion reaction was stopped by Fetal bovine serum (FBS, Corning) and the cells were centrifuged at 1200 rpm for 10 min. The pellet was resuspended and cultured in DMEM 10% FBS, supplemented with 1% penicillin/streptomycin and 2 mM L-glutamine (all purchased from Thermo Fischer Scientific).

HEK293 cells were transfected with the appropriate expression vectors using Lipofectamine 3000 reagent (Invitrogen) and selected in DCM containing 500 μg/mL Geneticin (G418) to generate stable cell lines HEK293-hLiTCo, HEK293-hLiTCo-Albu, HEK293-mLiTCo and HEK293-mLiTCo-Albu. Conditioned media were collected and purified using Strep-Tactin purification system (IBA Lifesciences) using an ÄKTA Go system (Cytiva). The purified antibodies were dialyzed overnight at 4°C against PBS Phosphate-Buffered Saline (PBS, Corning) with 150 mM NaCl at pH 7.0, analyzed by 12% SDS-PAGE under reducing conditions and stored at 4°C. Size exclusion chromatography was performed on a Superdex 200 Increase 10/300 GL column (Cytiva) on and AKTA-GO chromatography system (Cytiva). 50 μL of the solutions at 0.15 (mLiTCo) or 0.07 g/L (mLiTCo-Albu) were injected into the column and run at room temperature in PBS pH 7.4, with a flow rate of 0.5 mL/min.