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Protocols  >  Laboratory protocol  >  DCs differentiation and sti...

DCs differentiation and stimulation

Laboratory protocol

PBMC were isolated from buffy coats by density gradient centrifugation using Biocoll (BIOCHROM). Monocytes were isolated from PBMC using MACS anti-CD14 microbeads and a Midi-MACS® magnetic cell sorting device (both from Miltenyi Biotec, Bergisch-Gladbach,Germany). Cells were cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone). Differentiation of monocytes into dendritic cells was promoted by addition of granulocyte-macrophage colony-stimulating factor (GMCSF 1000U/ml, Chemicon) and recombinant IL-4 (1000U/ml, R&D Systems) for 5 days. DCs activation was induced by lipopolisaccharide (LPS 1microgram/ml, Sigma, St. Louis, MO), by Curdlan (100 microgram/ml, Wako), by R848 and yeast RNA and by yeast cells in different conditions of culture.

Protocol Steps:
  • Isolation of PBMC PBMC are isolated from buffy coats by density gradient centrifugation using Biocoll (BIOCHROM).
  • Monocyte isolation Monocytes are isolated from PBMC using MACS anti-CD14 microbeads and a Midi-MACS® magnetic cell sorting device (both from Miltenyi Biotec, Bergisch- Gladbach,Germany).
  • Cell culture Cells are cultured in RPMI 1640 medium (GIBCO BRL) supplemented with 2 mM L-glutamine (Sigma), 1% (vol/vol) non-essential amino acids, 100 mM sodium pyruvate, 50 U/ml of penicillin and 50 mg/ml of streptomycin (Gibco BRL) containing 10% (vol/vol) FCS (Hyclone).
  • Monocyte differentiation Differentiation of monocytes into dendritic cells is promoted by addition of granulocyte-macrophage colony-stimulating factor (GMCSF 1000U/ml, Chemicon) and recombinant IL-4 (1000U/ml, R&D Systems) for 5 days.
  • DC activation DCs activation is induced by lipopolisaccaride (LPS 1microgram/ml, Sigma, St. Louis, MO), by Curdlan (100 micrograms/ml, Wako), by R848 and yeast RNA and by yeast cells in different conditions of culture.
DCs differentiation and stimulation Graph


created over 8 years ago (2 March 2009)    last modified over 5 years ago (28 September 2011)   [ RDF Rdf ]   [ RelFinder Relfinder ]