Isolation of human macrophages and monocytes

Neutrophil isolation

Peripheral whole blood (18 ml) was taken from healthy volunteers and immediately laid under dual Percoll (GE Healthcare) density gradients of 1.098 and 1.079. Gradients were centrifuged at 150 x g for 8 min, followed by 1200 x g for 10 min. The neutrophil layer was removed to a fresh tube containing 3 volumes of red blood cell lysis buffer (0.83% NH₄Cl, 0.1% KHCO₃, 0.004% Na₂EDTA.2H₂O and 0.25% BSA) and gently agitated for 3 min to lyse contaminating red blood cells. Neutrophils were centrifuged at 400 x g for 6 min, and the resulting pellet washed twice in sterile endotoxin free PBS. Neutrophils were resuspended in serum free RPMI supplemented with 100 mM L-glutamine at a cell concentration of 1 x10⁵ cells/ml.

PBMC isolation 

PBMCs were isolated as described above, except the monocyte layer was extracted from the Percoll gradient. Monocytes were re-suspended in 1 mL adhesion media [500 mL RPMI 1640 with L-Glutamine (Gibco 21875-091), 5% heat inactivated (56⁰C for 30 min) pooled human AB serum (Sigma, Cat H4522-100ML), 5 mL Pen/strep (Sigma P4333)]. Monocytes were counted using a haemocytometer and trypan blue to determine viability. Cells were diluted to 2.5 x 10⁶ /mL in RPMI and 400 μL cells (0.5 x 10⁶ /mL) were seeded in 24-well plates. Cells were incubated at 37^oC, 5% CO₂ for 1h to allow adherence to the wells. Monocytes were washed three times with cold PBS to remove non-adhered cells.

Next, 1 mL differentiation media [500 mL RPMI 1640 with L-Glutamine (Gibco 21875-091), 10% heat inactivated (56 ⁰C for 30 min) pooled human AB serum (Sigma, Cat H4522-100ML), 5 mL Pen/strep (Sigma P4333, M-CSF 20 ng/mL (Sigma SRP3110)] was added to the wells. Cells were incubated at 37^oC, 5% CO₂ for three days. Media was removed and replaced with fresh differentiation media and incubated for another three days. On day seven, macrophages were ready for phagocytosis experiments.