Ficoll paque centrifugation

Patients’ peripheral blood samples were collected at baseline and after six months of GA in vivo administration.
Peripheral blood mononuclear cells (PBMC) were separated using Ficoll-Paque centrifugation (GE Healthcare); B cells were purified by autoMACS Pro Separator (Miltenyi biotec) using B cell Isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated cells, were analyzed using a FACS CANTO II (BD Biosciences, CA, USA) to confirm the purity of B cells (>98%). Cells at baseline were divided in two group. One underwent RNA extraction like B cells successively isolated from the same  MS patient after six months of GA. The second group was seeded (1 × 10⁶ cells/ml) with RPMI-1640 medium supplemented with 100 UI/ml penicillin, 100 μg/ml streptomycin (Life Technologies) and 5% autologous serum. Cultured cells were treated in vitro or not with 100 μg/ml GA for six hours in a humidified atmosphere containing 5% CO² at 37 C°. All experiments were carried out in duplicate.

Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque centrifugation (GE Healthcare, MA, USA). Total B-cells were isolated by using the Human B-Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany), from fresh PBMCs. The purity of B-cells was verified by flow cytometry using monoclonal anti-human CD19 fluorescein isothiocyanate (FITC). The percentage of memory B-cells was assessed by flow cytometry using monoclonal anti-human CD19 fluorescein, anti-human CD27 allophycocyanin and anti-human CD38 Pacific Blue antibodies (BD Biosciences, San Jose, CA, USA). The majority of B-cells were memory cells expressing CD19/CD27 and low CD38 [19 ,20 (link)].

The mouse MDSC cell line (MSC-2) was a kind donation from Gregoire Mignot. MSC-2 cells were cultured in RPMI 1640 media supplemented with 25 mM HEPES, 10% heat-inactivated fetal bovine serum (FBS), 1% antibiotic-antimycotic, and 1 mM sodium pyruvate. Patient-derived MDSCs were enriched from peripheral blood using the RosetteSep HLA-myeloid cell enrichment kit (Stemcell Technologies) followed by Ficoll-Paque centrifugation (GE healthcare). MDSC were isolated by subsequent negative selection of HLA-DRneg cells using anti-HLA-DR MicroBeads (Miltenyi Biotec) for 15 minutes at 4 °C and isolated using a MS-MACS column. Patient-derived MDSCs were acquired with informed consent under institutional review board (IRB)-approved protocols for human subject research at The Ohio State University, in accordance with the Declaration of Helsinki.

Lymphoblasts were purified from bone marrow or peripheral blood specimens using the Ficoll-Paque centrifugation method, according to the manufacturer’s instructions (GE Healthcare, Piscataway, NJ, USA). Genomic DNA was extracted from leukemic cells using standard phenol/chloroform-based methods. Briefly, 1 million cells were lysed in 10 mM Tris–HCl, 10 mM NaCl, 10 mM EDTA, 20 μg proteinase K, and 0.5% SDS by incubating at 37 °C for 16 h. Total RNA was further removed by adding 500 μg PureLink RNase A (Invitrogen, USA) and incubating for 10 min at 37 °C. An equal volume of phenol–chloroform–isopropanol (25:24:1) was added to lysates and mixed by shaking vigorously, followed by centrifugation at 16,100 × g at 4 °C for 5 min. The upper aqueous phase was transferred to a fresh tube; genomic DNA was then precipitated by adding 2× volume − 80 °C 100% ethanol. The DNA pellet was washed with 75% ethanol and rehydrated with Tris–EDTA buffer. The concentration of DNA was determined using a NanoDrop 1,000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA)^{32 (link)}.