Magellan v 5

The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) was used to assess metabolic activity of the PCLS as a readout for viability. Two hours post infection of equally sized PCLS, 60 µl of the MTS tetrazolium compound were added to each condition. Additionally, the MTS reagent was added to a PCLS maintained in 1% TritonX-100 as a negative control. The infected PCLS were incubated for further 2 h in the presence of the MTS tetrazolium compound. Four hours post infection, the purple formazan product was dissolved in culture medium by shaking the 24-well plate for 1 min. Subsequently, 100 µl of the supernatant was transferred in duplicates into a 96-well plate and the absorbance was measured at 492 nm using the TECAN SUNRISE Absorbance reader (Tecan, Salzburg, Austria) and analyzed with the Magellan V 5.0 software (Tecan, Salzburg, Austria).

Lactate Dehydrogenase (LDH) assay was performed with the Pierce™ LDH Cytotoxicity Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). After completing the PCLS generation, supernatant of PCLS was harvested in 24 h-intervals over 2 days and replaced with fresh 500 µl DMEM/F12 supplemented with 1% Penicillin-Streptomycin between two 24-h intervals. As positive control for LDH release, PCLS were treated with 1% TritonX-100 at 37°C, 5% CO₂, and 95% humidity for 45 min. The LDH assay was performed immediately after harvesting the supernatant according to the manufacturer’s instructions. The absorbance was measured at 492 nm and at a reference wavelength of 620 nm with the TECAN SUNRISE Absorbance reader (Tecan, Salzburg, Austria) and the Magellan V 5.0 software (Tecan, Salzburg, Austria).

The 2.5 × 10⁵ CD11c⁺ cells were stimulated as indicated in 96-well plates in 200 μl RPMI supplemented with 10% (v/v) FCS and P/S. Supernatants were harvested and analyzed for cytokines by commercially available enzyme-linked immunosorbent assay (ELISA) kits for TNFα and IL-12p40 (BD Biosciences, Heidelberg, Germany). Cytokines were detected by measuring the absorbance at 490 nm with a 650 nm reference in a photometer (Sunrise reader, Tecan, Salzburg, Austria). Cytokine concentrations were calculated according to a standard dilution of the respective recombinant cytokines using Magellan V 5.0 software (Tecan, Salzburg, Austria).

For the detection of the enzymatic activity of IDO, kynurenine was measured in the supernatants of iDCs and TLR-ligand-treated APCS after 3 days of culture. The protocol was adapted to the manufacturer’s instruction (Universität Ulm, Transplantationsforschung, Ulm, Germany). In brief, 150 µl cell culture supernatant was supplemented with 100 µl Trichloric acid (30%) for protein precipitation. After centrifugation, supernatant was transferred into 96-well plate format and incubated at 50°C for 30 min. For the measurement of kynurenine, a respective standard was used with the highest concentration of 50 µM, diluted stepwise 1:2. 150 µl color reagent was added to each well to start the reaction. After 5 min, absorbance was measured at 492 nm with a reference wavelength of 690 nm using a photometer (SUNRISE Absorbance reader, Tecan, Salzburg, Austria). Kynurenine concentrations were calculated with the Magellan V 5.0 software (Tecan, Salzburg, Austria).