Isolation of human peripheral blood monocytes and macrophage differentiation

Forty-five ml of blood samples from healthy adult donors were collected in complete RPMI 1640 (Cat# SH3025501, Cytiva, Marlborough, MA) (1% Sodium Pyruvate, 10% Fetal Bovine Serum and 1% Pen/Strep, GIBCO). Institutional review board at the University of Texas Health Science Center at Tyler (UTHSCT) approved these studies. Mononuclear cells were separated by Ficoll gradient method (1:3) by centrifuging cells at 500 x g (1600 rpm for 25 min). The cells were resuspended and washed twice with RPMI 1640, and then centrifuged at 400 x g (1350 rpm for 8 min). The monocytes were purified by positive selection using the CD14 MicroBeads (Miltenyi Biotech) according to manufacturer's recommendations. The cells were counted and resuspended in complete RPMI 1640 with 10% inactivated human serum. Cells were seeded on flacon 12-well plates (Fisher scientific) at a density of 0.5x10⁶ cells/well and incubated for 2 hours. After this time, the non-adherent cells were removed by soft mechanical lavage with complete RPMI 1640 containing 10% inactivated human serum inactivated. The monocytes were then cultivated in 2 ml of complete RPMI 1640 with 10% inactivated human serum supplemented with 5 ng/ml of GM-CSF, which was changed every third day until maturation and full differentiation of macrophages. Two to four wells were treated separately with complete RPMI 1640 with inactivated 10% human serum without the growth factor for comparative evaluation of monocyte differentiation with that of GM-CSF stimulated monocytes.