Antigen uptake, proteolysis and endosomal pH analyses
To assess antigen uptake, 5 x 104 purified cDCs were
incubated with 50 μg/mL OVA-Cy5 in complete medium for 0 – 90 minutes
at 37°C. cDCs were washed and the fluorescence measured by flow
cytometry. As a control, cDCs were pulsed with OVA-Cy5 for 90 min at
4°C. To assess antigen proteolysis and endosomal pH, 2.5 x
105 purified cDCs were incubated with 20 μg/mL DQ-OVA
(Life Technologies) or dextran-pHrodo for 15 min at 37°C. Cells were
washed, resuspended in complete RPMI and chased for the indicated time.
At each time point, cells were kept on ice and the fluorescence was
measured by flow cytometry. As a control, cDCs were pulsed with DQ-OVA
or dextran-pHrodo for 15 min at 4°C. The signal was calculated for the
37°C chase time point by subtracting the MFI at 4°C.