Human buffy coat blood units (n = 26; mean age of 44 ± 16.8; age range from 16 to 66 years old; 17 males and 9 females) were purchased from the New York Blood Center (New York, NY, USA, http://nybloodcenter.org/ ). Human buffy coats were added to 40 mL of chemical-defined serum-free culture X-VIVO 15TM medium (Lonza, Walkersville, MD, USA) and mixed with 10 mL pipette. Next, they were used for isolation of peripheral blood-derived mononuclear cells (PBMC). Mononuclear cells were isolated from buffy coats blood by Ficoll-PaqueTM PLUS (γ = 1.007, GE Healthcare, Chicago, IL, USA), followed by the removal of the red blood cells using Red Blood Cell Lysis buffer (eBioscience, San Diego, CA, USA). After three washes with saline, the whole PBMC were seeded in chemical-defined serum-free culture X-VIVO 15TM medium (Lonza, Walkersville, MD, USA), without adding any other growth factors, and incubated at 37 °C in 8% CO2 conditions.
To get the purified CD4+ T cells, PBMC were stained with CD4-FITC antibody for 30 min and purified with Anti-FITC Magnetic Beads (Miltenyi Biotech, Gladbach, Germany) according to the manufacturer’s instructions.
To get the purified CD4+ T cells, PBMC were stained with CD4-FITC antibody for 30 min and purified with Anti-FITC Magnetic Beads (Miltenyi Biotech, Gladbach, Germany) according to the manufacturer’s instructions.