Ficoll hypaque solution

The UCB obtained from the umbilical vein after the neonatal delivery of an infant was processed within 24 h for the isolation and separation of mononuclear cells (MNCs) with Ficoll-Hypaque solution (density = 1.077 g/cm³; GE Healthcare, Uppsala, Sweden), followed by previous protocol [20 (link), 21 (link)]. This protocol was approved by the Institutional Review Board of MEDIPOST Co., Ltd. (MP-2014-07-1-1). MNCs were washed with phosphate buffer saline (PBS) and cultured in minimum essential medium α medium (MEM-α, Gibco/Invitrogen, Carlsbad, Grand Island, NY, USA), supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere containing 5% CO₂. The culture medium was changed twice per week. The basic characterization of UCB-MSCs is summarized in Supplementary Table 1. Primary BM-MSCs and human AT-MSCs were purchased from Promega (Gibco/Invitrogen, Heidelberg, Germany). For growth kinetics, the trypan blue exclusion method was performed to analyze the expansion of live cells. At each passage (P), MSCs were cultured for 5 days, then reseeded at a cell density of 2,000 cells/cm². The PD at each passage was calculated by dividing the logarithm of 2 [20 (link)]. The analysis of PD was continued until the proliferation of cells was stopped.

Peripheral blood mononuclear cells (PBMCs) were separated from blood by gradient centrifugation over Ficoll-Hypaque solution (GE Healthcare Life Sciences, Pittsburgh, PA, USA). PBMCs (1×10⁷ cells/mL) were incubated with carboxyfluorescein succinimidyl ester (CFSE; Invitrogen/Molecular Probes, Eugene, OR, USA) at a final concentration of 0.5 μM for 10 min at 37°C. The cells were then washed with 10% FBS in RPMI two times in order to remove any excessive CFSE. The CFSE-labeled PBMCs (5×10⁵ cells/mL) were stimulated with or without immobilized anti-CD3 mAb clone OKT3 (Ortho Pharmaceuticals, Raritan, NJ, USA) (60 ng/mL) for 3 days in a 5% CO₂ incubator at 37°C. The cells were harvested and investigated for cell proliferation by monitoring the reduction in CFSE using FACSort flow cytometer (BD Biosciences).

The classification of SLE patients was based on the 2012 guidelines (10 (link)). All SLE subjects (n = 7, female, mean age 33 ± 13 years, SLE disease activity (SLEDAI) >10) and healthy controls (n = 12, male/female = 6/6, mean age 34 ± 9 years) were recruited from outpatient clinics or from among the medical staff in Shenzhen People’s Hospital (Shenzhen, China). No patient had been treated with an immune suppressant within the previous three months. The human sample studies and procedures were approved by the ethics committees of both Shenzhen People’s Hospital and Guangzhou Institutes of Biomedicine and Health (Guangzhou, China) (LL-KY-2019590) with informed written consent.
Eight milliliters of peripheral venous blood was drawn from both SLE patients and control subjects, followed by the addition of Ficoll–Hypaque solution (GE Healthcare, Switzerland) and density-gradient centrifugation. Red blood cell (RBC) lysis buffer was added to eliminate the remaining RBCs, and chilled PBS was used to wash the PBMCs. After quantification with a cell counting plate, PBMCs were stored on ice for further analysis.

This study was approved by the Institutional Review Board of MEDIPOST Co., Ltd. (MP-2014-07-1-1). UCB samples were obtained from the umbilical vein after delivery of the neonates. Mononuclear cells (MNCs) were separated from UCB using Ficoll–Hypaque solution (density = 1.077 g/cm³; GE Healthcare, Uppsala, Sweden). MNCs were washed and suspended in Minimum Essential Medium α (Gibco/Invitrogen, Carlsbad, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco). UCB-MSCs were cultured at 37 °C in a humidified atmosphere containing 5% CO₂, and the culture medium was changed twice a week [27 (link)]. Each UCB-MSC from three donors was used in our study (n = 3). Detailed information related to the UCB-MSCs is summarized in Supplemental Table S1. Results showed that pam3CSK4 and flagellin can induce TLR2 and TLR5 signaling pathway [28 (link)]. The pam3CSK4 and flagellin was obtained from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human GROa or IL-8 were purchased from R&D Systems (Minneapolis, MN, USA). The anti-TLR2 and anti-TLR5 antibodies used to block TLR2 and TLR5 stimulation, respectively, were purchased from Abcam (Cambridge, UK).