Anti mhc 1

RAW264.7 cells were harvested and preincubated with 0.5% BSA diluted in PBS for 30 min. The cells were then probed with fluorescence-conjugated anti-CD80, anti-CD86, anti-MHC-I, and anti-MHC-II (BD Biosciences) antibodies at 4 °C for 30 min. All Abs were diluted 100-fold before use. After washing with PBS, the expression of the corresponding molecules was measured by flow cytometry. The data were analyzed using FlowJo software.

To examine antigen uptake and processing, 1 μg/mL DQ-OVA (Molecular Probes), which is OVA conjugated to a self-quenched dye that emits green fluorescence upon degradation, was mixed with 400 μg/mL of an adjuvant and incubated for 16 h at 4 °C. DCs were plated onto μ-Slides (Ibidi GmbH) and stimulated with the mixture for 4 h at 37 °C followed by incubating with LysoTracker and DAPI (Molecular Probes). To examine the accumulation of MHC-I in acidic compartments, DCs were incubated with a mixture of OVA-Texas red (1 μg/mL; Molecular Probes) with either alum or PC nanogel for 3 h at 37 °C and then exposed to LysoTracker for the last 30 min. The cells were fixed, permeabilized and stained with an anti-MHC-I (BD) followed by Alexa488-conjugated anti-mouse IgG antibody (Invitrogen). Nuclei were counterstained with DAPI. The stained cells were examined with an LSM5 Pascal confocal fluorescence microscope (Carl Zeiss). The percentages of cells in which MHC-I colocalized with LysoTracker or OVA (Manders’ coefficient) in the insets were calculated using the JACoP plugin of the ImageJ software.

After the FhCL3 and LPS treatments as described above, the expression of surface molecules on DCs was quantified by flow cytometry using anti-CD11c, anti-MHCI, anti-MHCII, anti-CD40, anti-CD80, and anti-CD86, all purchased from (BD Bioscience, San Jose, CA, USA). For the exclusion of dead cells, we used the Zombie Aqua fixable viability kit (BioLegend, San Diego, CA, USA). Samples were collected using the FACSCanto II and FACS LSR Fortessa (BD Biosciences, San Jose, CA, USA) and data were analyzed using the Flow Jo Software.