Low endotoxin fetal bovine serum

Leukocyte concentrates obtained from healthy adult blood donors were kindly provided by the Dept. of Transfusion Medicine, University Hospital Schleswig-Holstein (UKSH) Campus Kiel. Informed consent was provided by all blood donors, and the use of leukocytes for research was approved by the Ethics Committee of the Medical Faculty (code D405/10). Monocytes were isolated from Ficoll-Hypaque separated PBMC by negative magnetic selection using the Pan Monocyte (order no. 130-096-537) or the Classical Monocyte (order no. 130-117-337) kits from Miltenyi Biotec (Bergisch Gladbach, Germany), or the EasySep Human Monocyte kit from StemCell Technologies (Cologne, Germany) according to the manufacturer’s protocols. Monocytes were differentiated for 6 days in the presence of 50 ng/mL M-CSF (ImmunoTools, Friesoythe, Germany). Fresh M-CSF was added after 4 days. Freshly isolated monocytes and M-CSF-differentiated macrophages were cultured at 10⁵ cells per well in round-bottom 96-well microtiter plates in RPMI 1640 medium (GIBCO) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 10% heat-inactivated low endotoxin fetal bovine serum (Bio&Sell, Feucht, Germany). In some experiments, monensin (3 μM) was added to monocytes cultures for 4 h before processing for intracellular IL-10 staining.

PBMC (4 × 10⁵ per well) or purified γδ T cells (0.5–1 × 10⁵ per well) with or without purified monocytes (1 × 10⁵ per well) were cultured in wells of 96-well round bottom microtiter plates (Nunc; Thermo Fisher Scientific, Waldham, MA, USA). Complete culture medium was RPMI 1640 (Thermo Fisher Scientific) supplemented with antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) and 10% of heat-inactivated low endotoxin fetal bovine serum (Bio&Sell, Feucht, Germany). Where indicated, cell cultures were supplemented with 10 ng/mL of IL-1β, 10 ng/mL of IL-12, 20 ng/mL of IL-18, or 5 µg/mL of neutralizing anti-cytokine antibodies. Vγ9Vδ2 T-cell lines were established from PBMC containing 2–3% CD3⁺Vδ2⁺ γδ T cells as described^{31 (link)}. Briefly, PBMC were stimulated with 2.5 μM zoledronate (Novartis) and 50 IU/mL IL-2. IL-2 was added every other day, and cultures were split on day 7 and day 9. After 12 days, such γδ T-cell lines contained > 90% CD3⁺Vδ2⁺ γδ T cells. γδ T-cell lines were seeded at 2 × 10⁵ cells per well in complete medium supplemented with 10 IU/mL IL-2. Cell cultures were stimulated or not with TLR ligands in the absence or additional presence of 1 nM HMBPP, and were incubated for 24 h at 37⁰C in a humidified atmosphere of 5% CO₂ in air. For measurement of intracellular cytokines, 3 μM monensin was added during the last 4 h to prevent cytokine secretion.