Ficoll density gradient centrifugation

PBMCs were isolated from HDs and RA patients by Ficoll-density gradient centrifugation (Axis-Shield). To obtain enough peripheral blood cells for the mAb blocking, cell signaling and lentiviral knockdown experiments, 400 ml peripheral blood was collected from other HDs with no inflammation or cancers. The cells were washed three times with sterile phosphate-buffered saline (PBS) and suspended at a concentration of 2 × 10⁶ cells/ml in RPMI-1640 (GIBCO) supplemented with 10% heat-inactivated fetal calf serum, 2 mM l-glutamine, and 1% penicillin–streptomycin (HyClone). CD4⁺ T cells were isolated from PBMCs using a human CD4⁺T-cell negative-isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. CD4⁺CD45RO⁺ (Tm) and CD4⁺CD45RO^- (Tn) cells were isolated using a CD45RO⁺cell isolation kit according to the manufacturer’s manual (Miltenyi Biotec). The cells were routinely analyzed by flow cytometry, and the purity of both the Tm and Tn population exceeded 90%.

Peripheral blood mononuclear cells (PBMCs) were isolated using a standard Ficoll procedure. Tissue-infiltrating leukocytes were dissociated from tissue specimens and collected as described previously [27 (link)]. Briefly, specimens were cut into small pieces and digested in RPMI 1640 (Gibco) supplemented with 100 μg/ml Liberase TL (Roche), 100 μg/ml DNase I (Sigma-Aldrich) and 20% FBS (Gibco) for 30 min at 37°C. The homogenates were filtered through a 150-mm cell strainer and separated by Ficoll density gradient centrifugation (Axis-Shield). The isolated PBMCs were washed with Hanks’ balanced salt solution and resuspended in RPMI 1640 supplemented with 10% FBS.

Myeloma BMSCs were isolated from remaining bone marrow samples of myeloma patients for routine diagnostic. Cells in the bone marrow sample were obtained by Ficoll density gradient centrifugation (AXIS-SHIELD). The cells were then plated in the tissue culture flasks at a concentration of 10⁶ cells/mL in Mesencult basal medium supplemented with MSC stimulatory supplements (both from Life technology). After 24 h incubation at 37°C in a 5% CO² humidified atmosphere, non-adherent cells were removed, and the adherent fraction was cultured in fresh medium. Cells used for future experiment were no more than 10 passages. Normal BMSCs cell line HS5 was obtained from ATCC, and cultured in Mesencult basal medium supplemented with MSC stimulatory supplements.