Cd1a pe

Human monocytes were isolated from mononuclear fractions of peripheral human blood by monocyte enrichment with (RossetteSep, Cat. no. 15028) for 20 min, then monocyte separation carried out by density Ficoll (GE Healthcare, Cat. no. 17-1440-03). Cells were cultured in the presence of GM-CSF (1000 unit/ml, Gemini Bio-Product, Cat. no. 300-124P) and IL-4 (1000 unit/ml, Gemini Bio-Product, Cat. no. 300-154P) at a concentration (3–4 × 105 cells/ml) for 5-6 days. Flow cytometry analyses were carried out to verify the immature DC phenotype (CD1a+, CD83−, CD14−, DC-SIGN+). Cell surface markers of DCs were evaluated by four-color immunofluorescence staining with the following antibodies: CD1a-PE (Miltenyi, Cat. no. 120-000-889), DC-SIGN-FITC (Miltenyi, Cat. no. 130-092-873), CD14-PerCP (Miltenyi, Cat. no. 130-094-969) and CD83-APC (Miltenyi, Cat. no. 130-094-186).

Human monocytes were isolated from mononuclear fractions of peripheral human blood by Human monocyte enrichment technique. After incubating the blood with the enrichment kit (RossetteSep, Cat. no. 15028) for 20 minutes, monocyte separation was carried out using medium density Ficoll (GE Healthcare, Cat. no. 17–1440–03). Cells were seeded in the presence of GM-CSF (1000 unit/ml, Gemini Bio-Product, Cat. no. 300–124P) and IL-4 (1000 unit/ml, Gemini Bio-Product, Cat. no. 300–154P) at a concentration (3–4 x 10⁵ cells/ml) for 5–6 days. Flow cytometry analyses were carried out to verify the immature DC phenotype (CD1a⁺, CD83^-, CD14^-, DC-SIGN⁺). Cell surface markers of DCs were evaluated by four-color immunofluorescence staining with the following antibodies: CD1a-PE (Miltenyi, Cat. no. 120–000–889), DC-SIGN-FITC (Miltenyi, Cat. no. 130–092–873), CD14-PerCP (Miltenyi, Cat. no. 130–094–969) and CD83-APC (Miltenyi, Cat. no. 130–094–186). After 30 min at 4°C and washing with staining buffer (PBS pH 7.2, 2 mM EDTA, and 2% FBS), cells were fixed in 1% paraformaldehyde. Positive marker expression was calculated as a percentage of total DCs by forward scatter and side scatter characteristics [14 (link),23 (link)].