Ficoll hypaque cushion

A total of 20 mL of peripheral blood was collected and used for the isolation of PBMCs by discontinuous gradient density centrifugation on a Ficoll-Hypaque cushion (Sigma, St. Louis, MO). Total RNA was extracted using the Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA concentrations and ratios were checked using a NanoDrop ND-1000 spectrophotometer (NanoDrop Products, Wilmington, DE), and the RNA integrity was assessed by microfluidic electrophoresis using a 2100 Bioanalyzer and RNA 6000 nanochips (Agilent Technologies, Santa Clara, CA). We used only samples that exhibited median RNA integrity number (RIN) ≥ 9.0.

Five-milliliter blood samples were withdrawn and added to an identical volume of PBS 1X (NaH₂PO₄ 1.9 mM, Na₂HPO₄ 8.1 mM, NaCl 154 mM). The mix was placed over a 5 mL Ficoll-Hypaque cushion (d = 1.077 g/mL) (Sigma-Aldrich, St. Louis, MO, USA) and centrifuged for 30 min at 800× g. The intermediate layer formed by mononuclear white cells was gently aspirated, washed three times in three volumes of HBSS (KCl 5.4 mM, Na₂HPO₄ 0.3 mM, KH₂PO₄ 0.4 mM, NaHCO₃ 4.2 mM, CaCl₂ 1.3 mM, MgCl₂ 0.5 mM, MgSO₄ 0.6 mM, NaCl 137 mM, D-glucose 5.6 mM, phenol red 0.02%) and centrifuged at 300× g for 10 min. The pellet containing cells was resuspended in 1 mL of PBS. Trypan blue testing and cell counting in a Neubauer chamber were performed afterwards. The number of collected cells ranged from 3 to 4 × 10⁶ cells.