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… Established cancer cell lines were cultured according to ATCC recommendations. DT22 cells and BM-MSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10%fetal bovine serum (FBS). MCF10A cells were cultured in DMEM/Ham’s F12 medium supplemented with 5% horse serum (Gibco BRL, Waltham, MA), insulin (10 μg/ml; Wisent Bioproducts, Saint-Jean Baptiste, QC), hydrocortisone (500 ng/ml; Sigma–Aldrich, St. Louis, MO), epidermal growth factor (EGF) (20 ng/ml; R&D, Minneapolis, MN), and cholera toxin (100 ng/ml; List Biological Laboratories, Campbell, CA).
Induction of myelin-reactive Th17 cells ex vivo and the adoptive transfer EAE model
… To induce EAE by adoptive transfer, CD4+ T cells were isolated from the MOG35-55-specific Th17 cell culture with the CD4+ T Cell Isolation Kit 5 days after stimulation. CD4+YFP+ T cells further purified on a FACSAriaTM III were adoptively transferred into Tcrb−/− mice via tail vein injection (5 × 106 cells/injection). One day after transfer, recipient mice were immunized with MOG35-55 peptide in CFA and then given an intraperitoneal injection of pertussis toxin (PTX, 500 ng/injection) (List Biological Laboratories, Campbell, CA, USA)12. …
Male MeCP2 WT and MUT mice, 9 weeks old, were anesthetized via i.p. with a mixture of xylazine and ketamine (16 mg/kg and 80 mg/kg respectively) and immunized subcutaneously at the right and left flanks with 200 μl of an emulsion containing 200 μg of myelin oligodendrocyte glycoprotein peptide (MOG35–55, NH2-MEVGWYRSPFSRVVHLYRNGK-COOH; synthesized at the Johns Hopkins University Synthesis & Sequencing Core Facility, Baltimore, MD, USA). The peptide was dissolved in sterile water at 2 mg/ml, mixed at a 1:1 ratio with complete Freund’s adjuvant (CFA, Sigma-Aldrich Co., St. Louis, MO, USA), supplemented with 4 mg/ml of Mycobacterium tuberculosis. Pertussis toxin (200 ng List Labs, USA) was dissolved in 100 μl of phosphate-buffered saline (PBS) and injected i.p. the same day of the immunization and 48 h later. …
Mice were immunized weekly for 6 weeks by oral gavage with 5mg ground blanched peanut (Western Mixers Produce & Nuts) and 10µg cholera toxin (CT) (List Biologicals) in 200µl of 0.2M sodium bicarbonate buffer per mouse …