Lymphocyte separation medium

The human BMMCs from healthy donors were isolated by density gradient centrifugation using lymphocyte separation medium (TBDscience). To analyze the effects of STC1 on monocytic differentiation, BMMCs were cultured with X-VIVO™ 15 Serum-free Hematopoietic Cell Medium (LONZA; 04-418Q) containing 50 ng/mL hSCF (R&D Systems; 255-SC-050) or/and 200 ng/mL rhSTC1 (R&D Systems; 9400-SO-050) in 5% CO₂ and humidified atmosphere at 37 °C for 3 days. To analyze the effect of STC1 on CD62L^hi CD8⁺ T_naïve cells, BMMCs were cultured with X-VIVO™ 15 Serum-free Hematopoietic Cell Medium (LONZA; 04-418Q) containing 200 ng/mL hIL-2 (R&D Systems; 202-IL-050) or/and 200 ng/mL rhSTC1 (R&D Systems; 9400-SO-050) in 5% CO₂ and humidified atmosphere at 37 °C for 3 days.

PBMCs were isolated from the blood of RA patients and healthy donors as previously described.^[20] Briefly, after gradient centrifugation by using lymphocyte separation medium (TBD science), mononuclear cells were collected, washed in RPMI 1640 medium (Gibco, ThermoFisher Scientific), and adjusted to 10⁶ cells mL⁻¹ in 1640 supplemented with 50 IU mL⁻¹ penicillin (Gibco, ThermoFisher Scientific), 50 µg mL⁻¹ streptomycin (Gibco, ThermoFisher Scientific), and 10% fetal bovine serum (FBS, Gibco, ThermoFisher Scientific). PBMCs (10⁶ cells mL⁻¹) were seeded in a 48‐well tissue culture plates (Corning, New York, USA), and then co‐incubated with anti‐CD3 (2 µg mL⁻¹) plus anti‐CD28 (2 µg mL⁻¹) antibody (biogems) with or without salivaricin A2 (12.5, 50, or 200 µg mL⁻¹) or salivaricin B (12.5, 50, or 200 µg mL⁻¹)^[12] at 37 °C in air with 5% CO₂. After 66 h culturing, the supernatants were collected, clarified by centrifugation (3000 rpm, 10 min, room temperature). Cytokines were measured by enzyme‐linked immunosorbent assay (ELISA) kits for IL‐10, IL‐17A, IL‐21, IFN‐γ, TNF‐α, and IL‐6 according to the manufacturer's instructions (Multisciences, Hangzhou, China). Informed consent was obtained from all subjects. This study was approved by Peking University People's Hospital Ethics Committee.

Chronically infected mice were randomly divided into 4 groups (18 mice in each group) and were treated with stress conditions for 0, 24, 48 or 72 hours respectively. At the conclusion of the treatments, mice were sacrificed and brain tissues were collected for cysts enrichment. To do that, homogenized brain tissues were subject to a density gradient centrifugation in Lymphocyte Separation Medium (TBD Science, China). In a 15 ml centrifugation tube, add 5 ml Lymphocyte Separation Medium and then add 5 ml tissue homogenates on top, the tubes were subsequently spun at 1000X g for 30min. The pellet fraction at the bottom with enriched cysts was collected for protein extraction. A trichloroacetic acid/acetone precipitation with phenol extraction method was used to isolate total proteins from enriched cysts, as previously described [37 (link)]. The concentrations of extracted proteins were measured by the Bradford protein assay before use.