Anti cd3 and anti cd28 monoclonal antibodies

The proliferation of CD8⁺ T cells upon co-culture with PC cells was detected using CFSE labelling as described previously [29 ]. Briefly, effector CD8⁺ T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies (BD Biosciences, San Jose, CA, USA). Then cells were labeled with CFSE (Invitrogen), and co-cultured with target PC cells at 37 ºC for 24 h. Cells after indicated treatment were harvested and the proliferation of CD8⁺ T cells was examined by flow cytometry on FACSCalibur (BD Biosciences, San Jose, CA, USA).

Jurkat T cells and human embryo kidney cell (HEK293T) were maintained in Dulbecco's minimum essential medium (DMEM, Gibco BRL, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, California, USA) and 1% penicillin/streptomycin. All cells were cultured in an incubator with 5% CO₂ at 37°C. Lipofectamine 2000 (Invitrogen) was used for miRNA transfection. Cells were assayed 48 h after transfection. Jurkat and human peripheral blood CD4⁺ T cells were activated, using anti-CD3 and anti-CD28 monoclonal antibodies (BD Biosciences, San Jose, CA, USA), as described previously [19 (link)].

Human hepatocyte cell line LO2 cells (Chinese Academy of Science cell bank, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified cell incubator with 5% CO2 at 37°C. FFA stock solution was prepared as previously described [8 (link)]. A mixture of oleic acid and palmitic acid (OA : PA = 2 : 1) was used to culture cells. Peripheral blood sample (30 ml) was donated by healthy donors. Peripheral blood mononuclear cells (PBMCs) were obtained using Ficoll-Histopaque (Sigma, St. Louis MO) density centrifugation, as previously described [9 (link)]. Anti-CD8 microbeads packed in Miltenyi MidiMACS columns (Miltenyi Biotec, Auburn, CA) were used to purify CD8+ T cells from PBMCs according to the manufacturer's instructions. CD8+ T cells were then cultured in RPMI-1640 medium with 10% fetal bovine serum and stimulated with anti-CD3 and anti-CD28 monoclonal antibodies (BD Biosciences, CA, USA).