Assessing the Antigen Processing Capacity of Cells Using DQ-Ovalbumin (DQ-OVA)

DQ-OVA is a self-quenched conjugate of ovalbumin which is strongly labeled with BODIPY dyes. It will exhibit bright green fluorescence upon proteolytic degradation into single, dye-labeled peptides. THP-1 derived M0 or iDC cells in Corning® 96 Well TC-Treated Microplates (Life Sciences, Tewksbury, Massachusetts, USA) with a volume of 100 μL per well using the same protocol as in the previous section on THP-1 cell Culture and Differentiation were incubated with medium containing 10 ug/mL DQ-OVA (Life Sciences, Tewksbury, Massachusetts, USA) for 1 h. After that, cells were washed twice with PBS without Ca²⁺ and Mg²⁺ (Gibco, Thermo Fisher, Waltham, Massachusetts, USA) and incubated with 100 μL of 0.25 % Trypsin-EDTA (Gibco, Thermo Fisher, Waltham, Massachusetts, USA) for 5 min. The cell suspension in Trypsin-EDTA was centrifuged for 5 min at 450 × g, after which the cell pellets were resuspended in 150 μL PBS with Ca²⁺ and Mg²⁺. The mean fluorescence intensity (MFI) of the FITC signal was recorded by flow cytometry of at least 5,000 cells for each sample using BD Accuri C6 software.