Lymphocyte separation medium

Fresh PBMCs were used after extraction for B cell isolation and all the analyses. PBMCs were isolated by lymphocyte separation medium (DAKEWE, Shenzhen, China) gradient centrifugation. B cell subsets were isolated by FACS Aria (Becton Dickinson, USA) based on their expression of CD19, CD24, and CD38 (Biolegend, San Diego, USA). The cell viability of B cell subsets was about 95% before sorting, and it turned to about 60% after sorting, using a TC20 automated cell counter (Bio-Rad Laboratories) and Trypan blue (Stemcell Technologies, Vancouver, Canada). CD19⁺ B cells and CD4⁺ T cells were also isolated by magnetic-bead purification with MACS kits (StemCell Vancouver, Canada).
For the preparation of thymic lymphocytes, fresh thymectomy tissue was immediately minced with scissors in PBS medium. Single-cell suspensions were obtained and washed by centrifugation at 4°C and 300 g for 5 min. Then, the thymic single cells were resuspended in RPMI 1640 (Gibco, Waltham, USA). Thymic lymphocytes were isolated by lymphocyte separation medium (DAKEWE, Shenzhen, China) gradient centrifugation.

To evaluate the treatment effect of dexamethasone sodium phosphate, a lymphocyte proliferation assay was performed by a previously published method [20 (link)]. In brief, the splenic lymphocytes were separated by a lymphocyte separation medium (Dakewe Biotech Co., Ltd., Shenzhen, China). The lymphocytes were seeded into plates at 5 × 10⁵ cells/well, and each splenic lymphocyte sample was plated in triplicate. Two micrograms of Concanavalin A (Sigma-Aldrich, Burlington, MA, USA) dissolved in 100 μL of RPMI-1640 medium was added as a positive control, and 100 μL of RPMI-1640 medium was added as a negative control. Subsequently, the lymphocytes were cultured at 37 °C with 5% CO₂ for 36 h, then the cell proliferative activity was measured by standard MTT assay as described previously [21 (link)]. The optical density (OD) was determined at wavelengths of 570 nm and 630 nm; the stimulation index (SI) was calculated with the following formula, SI = positive control (Concanavalin A) mean OD₅₇₀ − OD₆₃₀ / negative control (RPMI-1640) mean OD₅₇₀ − OD₆₃₀.

Heparinized blood samples from liver transplant recipients were collected at admission. The blood samples were centrifuged at 1800 rpm for 10 min at room temperature to separate plasma and blood cells. After transferring the supernatant, peripheral blood mononuclear cells (PBMCs) were obtained by density gradient centrifugation using the Lymphocyte Separation Medium (Cat: 7111011, DAKEWE). The plasma and PBMC were stored at -80°C until further use.