As 60193

BMDCs generated in the presence or absence of Ac₅3F_axNeu5Ac (250 µM) were washed in 1 × PBS and pulsed with OVA protein (Endograde, Hyglos GmbH, Germany) for 3 h or OVA H-2 Kb peptide (257–264, AS-60193, Tebu-bio) for 1 h at 37 °C. After the incubation, the BMDCs were washed three times, and 50.000 cells were seeded per well into a round bottom plate in culture medium. The CD8⁺ OT-I T cells were labeled with 3 µM CFSE according to the manufacturer’s instructions. 50.000 CFSE-labeled CD8⁺ OT-I T cells were added to the BMDCs (1:1 ratio) in culture medium. The co-cultures were incubated for 3 days at 37 °C in the presence of 0 or 10 ng/ml IL-2. After 3 days, the cells were analyzed by flow cytometry. Interferon gamma (IFNγ) levels in the supernatants were analyzed using an IFNγ ELISA (Thermo Fisher Scientific) following the manufacturer’s protocol.

For in vitro cross-presentation assays 80x10³ NSAID preincubated mBMDCs were pulsed with 80 μg/ml of endotoxin-free chicken egg ovalbumin (OVA protein, Endograde, Hyglos GmbH, Germany) in the presence of 400 ng/ml immune stimulatory complexes (ISCOMs). After pulsing, cells were washed and cultured overnight with 80x10³ B3Z cells. As a control for cell viability and/or MHC-I expression levels, DCs were pulsed with 5 ng/ml OVA K^b peptide (SIINFEKL, 257–264, AS-60193, Tebu-bio) 30 min before adding the B3Z cells. The presentation of OVA K^b peptide in H-2K^b results in production of β-galactosidase (LacZ) by B3Z cells, which can be detected by adding 0.15 mM chlorophenolred-h-D-galactopyranoside (Calbiochem), 9 mM MgCl₂, 0.125% NP40, and 7.5 mM DTT in PBS. Plates were incubated for 3 to 5 hr and absorbance values were measured at 595 nm using a photo spectrometer (Biorad).