Pe cy7 anti mhc 2 clone m5 114 15 2

De-immortalized DCs were seeded at 1*10⁵ cells/flask in 25 cm² cell culture flasks and allowed to settle overnight in a humidified atmosphere at 37 °C and 5% CO₂. Media were then removed and fresh media (5 ml) was given with 33% Cu-doped TiO₂ NPs at 40 µg/ml for 24 h. As controls, cells were exposed to the NPs in the media in the absence or presence of 1 µM MCC950 (InvivoGen). Media were removed, cells were washed with PBS, trypsinized and all supernatants, washing media and cells were added together and centrifuged at 2500 rpm for 6 min after which cells were stained with anti-CD86 antibody (FITC-anti-CD86 clone GL1, Thermo Fisher Scientific), anti-MHC II antibody (PE-Cy7-anti-MHC-II clone M5-114–15-2, Thermo Fisher Scientific) and anti-CD80 antibody (APC-anti-CD80 clone 16-10A1, Thermo Fisher Scientific) for 30 min at 4 °C. The cells were then centrifuged again, and resuspended in PBS after which they were run on the ImageStream X Mark II (Merck, Belgium) for analysis. Using iDEAS software, focused and single cells were selected and analysed for expression levels of the different markers.

RAW 264.7 macrophages were seeded at 1*10⁵ cells/flask in 25 cm² cell culture flasks and allowed to settle overnight in a humidified atmosphere at 37 °C and 5% CO₂. Media were then removed and fresh media (5 ml) was given with 33% Cu-doped TiO₂ NPs at 40 µg/ml for 24 h. Media were removed, cells were washed with PBS, trypsinized and all supernatants, washing media and cells were added together and centrifuged at 2500 rpm for 6 min after which the cells were split into 2 fractions and one half was stained with anti-CD163 antibody (SB436-anti-CD163 clone TNKUPJ, Thermo Fisher Scientific) and anti-CD206 antibody (APC-anti-206 clone MR6F3 Thermo Fisher Scientific) and the other half was stained with anti-CD86 antibody (FITC-anti-CD86 clone GL1, Thermo Fisher Scientific) and anti-MHC II antibody (PE-Cy7-anti-MHC-II clone M5-114-15-2, Thermo Fisher Scientific) for 30 min at 4 °C. The cells were then centrifuged again, and resuspended in PBS after which they were run on the ImageStream X Mark II (Merck, Belgium) for analysis. Using iDEAS software, focused and single cells were selected and analysed for expression levels of the different markers.