Fitc labeled ova ova fitc

Spleen cells (4 × 10⁶ cells) were incubated for 4 h with FITC-labeled OVA (OVA-FITC) (50 μg/ml, Thermo Fisher Scientific) in the presence or absence of GTS-21 (30 μM). To observe OVA-FITC uptake using confocal microscopy, the cells were plated onto poly-d-lysine-coated glass-bottom dishes, fixed with 4% paraformaldehyde for 20 min at 4°C, and permeabilized and blocked with Blocking One (Nacalai tesque) containing 0.5% Triton X-100 for 1 h at room temperature. The cells were then incubated with PE-conjugated anti-CD11b and APC-conjugated anti-CD11c Abs for 20 min at 4°C. Nuclei were stained with DAPI (300 nM) for 10 min at room temperature. Cells were imaged using a confocal microscope (Zeiss LSM 800 Meta, Carl Zeiss, Inc., Germany) equipped with an oil-immersion objective (40 × , NA = 1.3). Fluorescence images were processed using ImageJ 1.37a (National Institutes of Health). For flow cytometric analysis, spleen cells were stained with PE-conjugated anti-CD11b and APC-conjugated anti-CD11c Abs for 20 min at 4°C and subjected to flow cytometry.

IT macrophages (1.0 × 10⁶/mL) were incubated with FITC-labeled OVA (OVA-FITC; 500 ng/mL) (Thermo Fisher Scientific) in RPMI complete medium at 37 °C for 2 h. The cells were then washed with PBS containing 2% FBS (PBS/2% FBS) and analyzed by flow cy-tometry. The fluorescence signal (mean fluorescence intensity; MFI) originating from intracellularly captured OVA-FITC was used to assess the antigen uptake ability in macrophages.