MUTZ-3 Dendritic Cell Maturation

The human MUTZ-3 cell line was purchased from DSMZ (Germany). The cells were grown in α-MEM with 20% heat-inactivated FBS (PAA Laboratories-GE Healthcare Life Sciences), 1% Glutamax (Life Technologies) and 40 ng/ml GM-CSF (CellGro) as previously described (Nelissen et al., 2009 (link); Song et al., 2015 (link)). The MUTZ-3 cells were differentiated to immature DC with 62.5 ng/ml GM-CSF, 100 ng/ml IL-4 (CellGro) and 2.5 ng/ml TNF-α (CellGro) for 4 days (Zavašnik-Bergant and Bergant Marušič, 2016 (link)). The viability of the cells was evaluated using Trypan Blue (Sigma-Aldrich). The differentiated MUTZ-3 cells were matured with 20 ng/ml LPS (Sigma-Aldrich) for 24 h. Alternatively, the MUTZ-3 cells were stimulated with LPS in the presence of tick cystatin OmC2 (0.4 or 0.8 μM). Cystatin solution was filtered through a 0.22 μM Durapore PVDF membrane/Millex GV filter unit (Millipore). In control experiments, sterile PBS was added instead of cystatin OmC2, and the cells were cultured for 24 h. In addition, immature DC were matured with LPS in the presence of 0.4 or 0.8 μM human cystatin C (Zavašnik-Bergant et al., 2005 (link)).