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发表于 2015-6-29 23:08:43
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这方面实验我没做过，但看了下相关文献，感觉应该还是诱导条件的问题：
例如《Induction of Th17 Lymphocytes and Treg Cells by Monocyte-Derived Dendritic Cells in Patients with Rheumatoid Arthritis and Systemic Lupus Erythematosus》中的相关内容可供参考：
2.3. Generation of Monocyte-Derived Dendritic Cells (mo-DC)
Monocytes (1 × 106/mL) were plated in RPMI-1640 culture medium supplemented with 10% FCS, 2.0 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin (Gibco BRL, Grand Island, NY), 1% sodium pyruvate (Gibco), 1% nonessential amino acids (Hyclone Laboratories, South Logan, UT), and 50 mM 2-mercaptoethanol (Gibco) in the presence of 200 ng/mL rhGM-CSF and 15 ng/mL rhIL-4 (eBiosciences) at 37°C and 5% of CO2. Cells were fed on days 2 and 4 by changing one half of the medium and keeping the same concentration of IL-4 and GM-CSF. At day 6, DCs were induced to mature by adding 200 ng/mL of LPS (Sigma-Aldrich). Forty-eight hours after stimulation, supernatants were collected for cytokine measurement. In the case of the induction of tolerogenic dendritic cells, the differentiation of monocytes was carried out in 24-well tissue culture plates previously coated with recombinant human P-selectin (10 μg/mL) or PD1 (2.5 μg/mL) (BioLegend, San Diego, CA) by overnight incubation at 4°C. In other set of experiments, monocytes were induced to differentiate in the presence of IL-10 (40 ng/mL, Biolegend).
2.4. Lymphocyte-DC CoCultures and Induction of Th17 and Treg Differentiation
For the induction of differentiation of Th17 cells, mature mo-DC and autologous CD4+ T cells were cocultured in 24-well plates at 1 : 10 ratio in complete Iscove’s modified Dulbecco medium (IMDM, Gibco BRL). Plates were previously coated with 10 μg/mL anti-CD3 (eBiosciences) and anti-CD28 (Immunotech) mAbs, and Th17 differentiation was induced by adding rhIL-23 (10 ng/mL, R&D systems, Minneapolis, MN), IL-6 (50 ng/mL), IL-1β (10 ng/mL), IL-21 (50 ng/mL), TNF-α (10 ng/mL, all from eBiosciences), and anti-IL-4 and anti-IFN-γ mAbs (5 μg/mL, BioLegend). Under such conditions, cells were cultured during 5 days at 37°C and 5% of CO2, and then the percent of Th1 and Th1/Th17 cells was determined by flow cytometry. In the case of the induction of Th1/Th17 cells, no anti-IFN-γ antibody was added. For induction of Treg cell differentiation, DCs generated in the presence of tolerogenic stimuli (IL-10, PD1, P-selectin) were cocultured with autologous CD4+ T cells for five days, and then cells were immunostained for CD4, CD25, and Foxp3, and they were analyzed by flow cytometry.
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发表于 2015-6-29 21:57:53
