Polymorphprep

Polymorphonuclear granulocytes (PMN) and peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of healthy donors by density gradient centrifugation using either Polymorphprep^® (Progen, Heidelberg, DE) or Ficoll Paque Plus (GE Healthcare, Chicago, IL, USA), respectively, as previously described (19 (link), 20 (link)). PBMC were then used for generation of non-polarized (M0) macrophages as described (17 (link)). Briefly, after incubation in monocyte attachment medium (PromoCell, Heidelberg, DE) for 30 min at 37°C, cells were washed three times with PBS to dispose non-adherent cells and resuspended in X-VIVO 15 medium (Lonza, Basel, CH) supplemented with 0.5% v/v penicillin/streptomycin (Gibco, Amarillo, TX, USA). After culturing for 24 h, 50 ng/ml M-CSF (PeproTech, Rocky Hill, CA, USA) were added and refreshed every 72 h at least twice before macrophages were used in ADCP experiments.

Neutrophils were isolated from fresh blood, as previously described [43 (link)]. In short, fresh blood was drawn from healthy volunteers by a physician and directly used for isolation. Blood was layered 1:1 on Polymorphprep solution (Progen) in a 50 mL falcon tube and centrifuged at 472× g for 30 min at RT without brake. Afterward, the monocyte and plasma layer was removed, and with a fresh plastic Pasteur pipette, the neutrophil layer was transferred to a new falcon tube, directly filled up with 1× PBS at RT, and centrifuged at 472× g for 10 min with brake. The cell pellet was treated with 5 mL sterile H₂O for erythrocyte lysis for 15 s and immediately filled up with 1× PBS, maximum 2 times. After another round of centrifugation at the same settings, the cell pellet was resuspended in 1 mL RPMI at RT. Cell count was determined as stated above.

Human neutrophils were isolated from human blood using PolymorphPrep™ (PROGEN Biotechnik GmbH, Heidelberg) according to the instructions of the manufacturer. The neutrophils were suspended in RPMI 1640 (Invitrogen, Thermo Fisher Scientific, Darmstadt, Germany). Bacteria were grown over night and washed with PBS. For opsonization 10⁷ CFU/ml were incubated for 20 min with 10% human serum at room temperature. The opsonized bacteria were incubated with 10⁷ human neutrophils/ml (1:1) and 5% serum for another 30 min at 37 °C. As a reference, a sample of the opsonized bacteria was incubated without neutrophils. To determine the survival rate, the samples were centrifuged and the pellets lysed in sterile distilled water. The counts of viable GAS were determined following serial dilution and plating on THY agar. To determine the proportion of extracellular bacteria, the samples were centrifuged for 5 min at 100 g for separation. The supernatant was collected, centrifuged at 13.000 g, and the bacterial pellet was dissolved in PBS.

All reagents were filtered through Detoxi-Gel Endotoxin Removing Gel Columns (Thermo Fisher Scientific) prior to neutrophil isolation. Human neutrophils were isolated using a density-gradient separation method (Ostrowski et al., 2020 (link)). Briefly, 30 ml of blood was collected in sterile BD Vacutainer EDTA (BD Biosciences, Mississauga, ON, Canada) tubes and layered on the top of PolymorphPrep (Progen, Wayne, PA, USA) in a 1:1 ratio. The layered mixture was spun at 500 g (acceleration 1 and deceleration 0) for 30 min to separate polymorphonuclear neutrophils (PMN) from mononuclear cells. PMN were washed with Hank’s balanced salt solution with calcium and magnesium (HBSS+/+; Wisent) and then subjected to red blood cell lysis using 0.2% NaCl followed by 1.6% NaCl for 30 s each. Neutrophils were resuspended in HBSS+/+ and used within 1 hr for all experiments.