1640 glutamax

Dendritic cell and macrophage cultures were started from BM cells, which were isolated from murine femurs and tibiae. iCD103 DCs were generated as described previously (47 (link)). In brief, BM cells were cultured in complete RPMI (cRPMI) 1640 GlutaMAX medium (Thermo Fisher Scientific), supplemented with 10% heat-inactivated FCS (Biochrom), 500 U penicillin-streptomycin (PAA laboratories), and 50 μM β-mercaptoethanol (Thermo Fisher Scientific) with a combination of GM-CSF and FLT3L (both self-made) for 9 days, followed by re-plating with both growth factors for additional 7 days at 37°C with 5% CO₂. For generating GM-CSF DCs, BM cells were cultured in cRPMI supplemented with 5% culture supernatant of a GM-CSF-producing cell line (48 (link)). For generating BMDMs, BM cells were incubated in cRPMI supplemented with L929 cell conditioned medium (LCCM; self-made) as a source of murine M-CSF for 7 days. After 3 days, half of the medium was replenished by fresh medium containing LCCM. Flt3-L-producing CHO Flt3-L FLAG cells were generated by Dr. Nicos Nicola and kindly provided by Dr. Karen Murphy (WEHI, Melbourne, VIC, Australia). The LCCM producing L929 cell line was kindly provided by Dr. Roland Lang (Universitätsklinikum Erlangen, Erlangen, Germany).

A. fumigatus conidia from glycerol stocks frozen at −80 °C were spread onto Sabouraud dextrose agar in T75 culture flasks (Greiner Bio-One, Frickenhausen, Germany), and incubated at 37 °C. After 7 days, conidia were harvested via immersion in Phosphate Buffered Saline (PBS) containing 0.05% Tween-80 (Thermo Fisher Scientific, Renfrew, UK). Suspensions were passed through a sterile 40 µm strainer to remove hyphae and washed twice using PBS. Suspensions were counted using a Neubauer improved haemocytometer. Conidial suspensions were diluted in RPMI (1640 + Glutamax; Thermo Fisher Scientific).

Cryovials with PBMC were thawed in a 37°C water bath and transferred to 20 ml of RPMI medium 1640 + GlutaMAX (RPMI), (Gibco, MA USA). The PBMC was collected by centrifugation at 200 × g for 10 min, washed with 40 ml of RPMI, and suspended in 1 ml of RPMI + 10% HS + 10 µg/ml Gentamicin (complete RPMI). After resting at 37°C in 5% CO₂ for 2 h, the PBMCs were counted and diluted in complete RPMI. For FACS analysis, 8 × 10⁵ cells were seeded per well in a round-bottom 96-well plate (Sarstedt, Nümbrecht, Germany). For the Lymphocyte proliferation assay (LPA), 2 × 10⁵ cells were seeded per well. To some wells, Ft antigen (2.5 µg/ml), prepared from F. tularensis LVS as described previously (20 (link)) or Concavalin A (ConA) (2.5 µg/ml), was added (stimulated cells) whereas other wells contained complete RPMI only (resting cells). After 3 days of incubation, 300,000–500,000 cells per sample were collected and analyzed by FACS. From separate wells, supernatants were transferred to a 96-well plate and stored at −80°C until analyzed for cytokine content by multiplex cytokine analysis. For LPA, tritium-thymidine (0.003 mCi/ml) (Perkin Elmer MA USA) was added to the cells, and after 6 h, incorporated thymidine was measured using a 1450 microbeta liquid scintillation & luminescence counter (Trilux Chelmsford UK). All time points from two to three individuals were included in each experiment.

Cell lines were obtained from the American Type Culture Collection (ATCC) and cultured according to standard mammalian tissue culture protocols and sterile technique. The murine RAW-264.7 macrophages (ATCC #TIB-71) were cultured in DMEM (Gibco #41966), CT26 cells from mice (ATCC #CRL-2638) were cultured in RPMI medium 1640 GlutaMAX (Gibco #61870044). All media was supplemented with 10% FBS and 1% Penicillin-Streptomycin. All cells were tested for mycoplasma before performing the experiments using the MycoAlert mycoplasma detection kit (Lonza #LT27-221) following manufacturer’s instructions. For M1 polarization of RAW-264.7 cells, 300.000 cells/well were seeded in a 6-well plate with 100 ng/mL of IFN-γ (Biolegend #575304) and for M2 polarization, cells were co-cultured with 100 ng/mL of IL-4 (Biolegend #574304). Hypoxia cultures were performed at 1% oxygen and 5% CO₂ in an In Vivo2 400 hypoxic station (Ruskinn Technologies).