Ficoll hypaque

PBMCs were isolated from the peripheral blood of healthy blood donors by Ficoll-Hypaque (Solarbio, Beijing, China) density gradient centrifugation with informed consent at Anhui Blood Center. PBMCs were cultured in RPMI medium supplemented with 10% FBS in the presence of 50 IU/mL rhIL-2, 20 nM scFvCD16A-sc4-1BBL, 20 nM scFvCD16A, 60 nM mn4-1BBL, or 20 nM scFvCD16A + 60 nM mn4-1BBL for 24 h. The cells were collected and analysed by flow cytometry according to a previously described method [71 (link)].

Ten milliliters of EDTA-anticoagulant peripheral blood samples were collected from each enrolled subject, and plasma samples were harvested by centrifugation at 3000×g for 10 min. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll–Hypaque (Solarbio, Beijing, China) density gradient centrifugation. Approximately 10⁷ of PBMCs could be isolated from 10 ml of peripheral blood.

Human CD4⁺IL-10⁺ T cells were induced in vitro as per previously described protocols [21 (link)]. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy donor blood samples (Central Blood Bank in Yantai, China) using gradient separation with Ficoll-Hypaque (1.077 g/mL; Solarbio, Beijing, China). PBMCs were cultured using the RPMI-1640 medium (HyClone, Boston, MA) and incubated at 37°C, and naive CD4⁺ T cells were separated using a naive CD4⁺ T cell isolation kit (human; Miltenyi Biotec, San Diego, CA) according to the manufacturer’s instructions.
Naive CD4⁺ T cells were treated with or without the Nrf2 inhibitor-ML385 (10 μM, MCE, NJ, USA) for 12 h, and then naive CD4⁺ T cells were cultured with or without hPMSCs. Anti-human CD3ε and anti-human CD28 mAb (1 μg/mL, Life Technologies, Waltham, MA, USA), recombinant human (rh) IL-2 (200 U/mL, Proteintech, Wuhan, China), IL-10 (100 U/mL, Proteintech), IL-27 (63 U/μL, Proteintech), and IFN-α2b (2.09 U/μL, PBL Assay Science, NJ, USA) were added to the coculture system. After 3 days, the cells were stimulated using PMA/ionomycin (1.25 μg/mL, 0.25 mg/mL, MultiSciences, Hangzhou, China) and BFA/monensin (0.75 mg/mL, 0.25 mg/mL, MultiSciences) for 5 h and subjected to surface staining and intracellular staining procedures.