Heat inactivated fetal calf serum

For the analysis of the IL-22 concentration in the sera of naïve and infected mice, serum was prepared using the BD Microtainer SST Tubes (BD Pharmingen) and the IL-22 concentration was determined by cytometric bead array according to the manufacturer’s instructions (Biolegend).
To analyze the cytokine secretion of spleen cells, single cell suspensions were prepared at the indicated time points after infection and 1 × 10⁶ cells were incubated in 300 μl Iscove’s Medium (Biochrom), supplemented with 2 mM L-glutamine, 10% heat-inactivated fetal calf serum (Biochrom), penicillin and streptomycin (100 U/ml and 100 μg/ml, respectively; Biochrom) for 24 h. The concentration of the indicated cytokines in the supernatant was determined by cytometric bead array according to the manufacturer’s instructions (BD Bioscience). Cytokine concentrations were analyzed using the FCS Filter and FCAP Array software (Soft Flow).

Bone marrow-derived dendritic cells (BMDCs) were generated by cultivation of bone marrow progenitor cells from C57BL/6 WT and Cd1d^−/− mice in complete medium consisting of 10% heat-inactivated fetal calf serum (Biochrom), 1% L-glutamine, 1% penicillin, 1% streptomycin, 0.1% Mercaptoethanol (all from Gibco) in RPMI (Gibco) supplemented with 200 ng/ml GM-CSF (PeproTech). After 7 days of culture, BMDCs were harvested.
For stimulation BMDCs (2 × 10⁵) from WT and cd1d^−/− were cultivated in the presence of either APE (1:10 dilution originating from 30 mg/ml of pollen) or 100 ng/ml of highly purified and lipopeptide-free S. friedenau LPS (kindly provided by Prof. Helmut Brade, Research Center Borstel, Germany) in round bottom 96-well-plates for 24 h at 37°C in 5% CO₂. Maturation was assessed by the surface up-regulation of CD40, CD80, MHC class-II, and CD1d. Cells were labeled with anti-mouse CD80 (clone 16-10A1), CD1d (clone 1B1), CD40 (clone 3/23), CD11c (clone N418), MHC class-II (clone M5114.15.2) and washed in FACS-buffer containing 2 μg/ml PI. PI-negative cells were analyzed by flow cytometry.

Peripheral blood mononuclear cells were isolated from buffy coats derived from blood donation bottles of six healthy volunteers by density centrifugation on Ficoll density gradient (Biochrom AG, Berlin, Germany) as previously described (Spiliopoulou et al., 2012 (link)). Briefly, collected mononuclear cells were washed in PBS and resuspended in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (Biochrom AG) and 2 mM L-glutamine (HyClone), (CM). Cells at a density of 1 × 10⁶ cells/mL per well, were then seeded in 24-well flat bottom tissue culture plates (Sarstedt, Nümbrecht, Germany) and cultured at 37°C in a humidified, 5% CO₂ atmosphere.

The murine leukemic macrophage cell line RAW264.7 (ATCC^® #TIB-71™), the human epithelial cervical adenocarcinoma cell line HeLa (ATCC^® #CCL-2™) and the virus packaging cell line HEK293TT (established from primary embryonal human kidney cells transformed with modified human adenovirus) [47 (link)] were grown and maintained in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal calf serum (Biochrom, Cambridge, UK), 100 U/ml penicillin and 100μg/ml streptomycin (complete DMEM). All cells were grown at 37 °C in 5% CO₂/95% air humidified atmosphere.