Human peripheral blood dendritic cells induced culture and identification of surface markers.docVIP

 PAGE \* MERGEFORMAT 6 Human peripheral blood dendritic cells induced culture and identification of surface markers [Keywords:] Dendritic cells [Keywords:] dendritic cell; peripheral blood; co-stimulatory molecule 0 Introduction Dendritic cells (dendritic cells, DC) of the features and functions of tumor immunity is currently a hot research, this study will be DC’s culture and surface markers, etc. for further study, in order to lay the foundation for further study of DC. 1 Materials and methods 1.1 Materials Recombinant human granulocyte-macrophage colony-stimulating factor (rhGMCSF), recombinant human interleukin 4 (IL4), tumor necrosis factor-α (TNFα ) were purchased from Shenzhen Crystal US companies, FITC labeled monoclonal antibodies: CD83, CD40, HLADR, CD86 were purchased from Shenzhen Crystal US companies. lymphocyte separation medium (Shanghai Reagent Plant), fetal calf serum (Hangzhou Sijiqing bio-engineering company). Instrument: The United States B2D’s FACS Calibur flow cytometer, Nikon fluorescence microscope. 1.2 Methods At room temperature to 25 mL of blood-rich composition and equal amount of white blood lymphocyte separation medium mixing, using FicollHypaqu density gradient centrifugation at room temperature for 3000 r / min, centrifugation 20 min after the collection of mononuclear cells. Regulate cell concentration of 106/mL , and then posted on walls in 6-well plates, each hole plus 2 mL, 3 h after the suspension cells absorb and use 37 ℃ pre-heating fresh medium gently wash away the non-adherent cells, to join No. Ⅰ nutrient solution: 100 mL / L fetal calf serum RPMI 1640 containing GMCSF 50 ng / mL, IL4 10 ng / mL, at 37 ℃ , 50 mL / L CO2 incubator box culture, changing the amount of liquid a day and a half times, 3 times for fluids Ⅱ No. nutrient solution: 100 mL / L fetal calf serum RPMI 1640 containing GMCSF 50 ng / mL, IL4 10 ng / mL, TNFα 20 ng / mL, is still fluid a day and a half times the amount of change. cell cul