Cells were washed once with Hanks’s balanced salt solution and cu

Cells were washed once with Hanks’s balanced salt solution and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA) supplemented with 5% fetal

calf serum (Gibco, Paisley, UK), 1% L-glutamine (Sigma, St Louis, MO, USA), 1% non-essential amino acids (Sigma), 2 × 10−5 M 2-mercaptoethanol (Amresco, Solon, OH, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). All cells were adjusted to 2 × 106 cells/ml. MNC suspensions (4 × 105) obtained above were seeded in triplicate in 96-well, round-bottomed microtitre plates at different lymphocyte : astrocyte ratios (10:1, 1:1 and 1:5). Cells were stimulated with 25 μg/ml MOG35–55 peptide for Ibrutinib ic50 72 h. For anti-CD3/CD28-induced

cell proliferation, 96-well culture plates were coated with purified anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) (5 μg/ml each; eBioscience, Ltd, Ireland, UK). ConA (Sigma, St Louis, MO, NVP-AUY922 nmr USA) was used at 5 μg/ml. Proliferation was measured by [3H]-thymidine (specific activity, 60 μCi/mmol; Institute of Atomic Energy, China; 0·5 μCi/well) incorporation after 72 h in complete DMEM medium. Astrocytes were cultured at a concentration of 1 × 106 cells/well in 12-well plates, then incubated with 2 μg/ml goat anti-mouse-IL-27 antibody (R&D Systems, Minneapolis, MN, USA) [37] or isotype control immunoglobulin (Ig)G2a in 2 ml medium for 12 h to neutralize IL-27. ifoxetine Astrocytes were co-cultured with MNCs (1 × 107) harvested from the lymph nodes of EAE mice in 2 ml lymphocyte culture medium. The cells were incubated at 37°C, 5% CO2 for 72 h. Supernatants were collected for measurement of the levels of soluble cytokines. Astrocytes (1 × 106) were co-cultured with lymph node lymphocytes (1 × 107) harvested from 7 dpi mice in 2 ml lymphocyte culture medium. Where indicated, lymphocytes were also seeded in Transwell™ insert (24-well plates, 3 μm pore size;

Corning, NY, USA). Twenty-five μg/ml MOG35–55 peptide was incubated as antigen and the supernatants were collected 72 h later. Measurement of cytokine levels in cell culture supernatants was performed by enzyme-linked immunosorbent assay (ELISA) using commercially available ELISA kits, in accordance with the manufacturer’s instructions. IFN-γ, IL-17 and IL-4 ELISA kits were purchased from Peprotech (Rocky Hill, NJ, USA). The TGF-β ELISA kit was obtained from Boster, China. Results are expressed as pg/ml. Total RNA was prepared from spinal cords or lymph node MNCs using TRIzol reagent (Invitrogen). cDNA was synthesized using a reverse transcription–polymerase chain reaction (RT–PCR) kit from TaKaRa (Kyoto, Japan). RT–PCR was used to detect MHC-II genes using the following forward 5′-GATCGGATCCAACCCTGCTGAGGATTCA-3′ and reverse 5′-GATCGGATCCTGTCCTCGGCTGGGAAGA-3′ primers.

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