Ow cytometer

Cell Counting Analysis Kit (CCK-8, Beyotime, China) for cell viability detection was performed according to the manufacturer's instructions. Brie y RAW 264.7 cells were seeded in a 96-well plate at a density of 5×10 3 /well . After 24h, the medium was replaced with fresh α-MEM medium containing AGEs at concentrations of 0, 100, 200, 400 mg/L for 12, 24, 36, and 48 h. Then, after the supernatant was discarded and cells were washed with PBS twice, 10μL CCK-8 solution was added to each well and cultured at 37 °C for 3 h. The absorbance was measured by enzyme labeling instrument (PerkinElmer, USA) at 450 nm wavelength.
An Annexin V-FITC apoptosis assay kit (Beyotime, China) was used to estimate the apoptosis rate of RAW264.7 cells. Brie y, cells in logarithmic growth phase were inoculated in 6-well plates and cultured for 24 h. Then the medium was replaced with fresh α-MEM medium containing AGEs at concentrations of 0, 100, 200, 400 mg/L for 24 h. After washed with PBS twice, cells were stained with Annexin V-FITC and propidium iodide (PI) in binding buffer at 4˚C. Samples were detected with ow cytometer (BD In ux, USA) and the results were statistically analyzed by FlowJo 7.6.1.

For further exploring the endocytic pathways of DCs to NaVs, we used several uptake inhibitors to pretreat cells separately to block the corresponding endocytic pathway. Brie y, BMDCs were co-cultured with fucoidan (FCD, 10 µg/mL), chlorpromazine (CPM, 10 µg/mL) or methyl-β-cyclodextrin (MBCD, 10 µg/mL) for 1 hour, respectively. Afterwards, the pretreated cells were co-incubated with free FITC-OVA or NaVs (containing FITC-OVA) for hours while untreated cells were used as negative control. Then the quantity of FITC-OVA in BMDCs was detected using a ow cytometer (BD Biosciences, CA, USA) and the reduction of antigens uptake of NaVs compared to negative control group was calculated.

ROS and mitochondrial ROS (mitoROS) levels were measured using a Reactive Oxygen Species Assay Kit (Beyotime, China) and MitoSOX Red reagent (Invitrogen, USA), respectively, according to the manufacturer's instructions. Brie y, 2 × 10 5 KFb were seeded into 6-well plates and cultured in complete culture medium containing different concentrations of LY294002 (0, 5, and 25 µM) in incubators under hypoxia or normoxia at 37°C for 24 h. For ROS detection, KFb were harvested with 0.25% trypsin and incubated with 10 µM DCFH-DA working solution for 20 min at 37°C in the dark. For mitoROS investigation, cells were incubated in 1 ml of 5 µM MitoSOX reagent working solution at 37°C in the dark. The cells were then washed twice with DMEM without FBS to remove the working solution that did not enter the cells. Finally, the uorescence intensity was assessed using a ow cytometer (BD, USA).

Annexin V apoptosis detection kits (Bio-Vision, SF) was used to quantify cells apoptosis according to the manufacturer's instruction. Cells were stained with FITC-conjugated annexin-V in the dark at room temperature and were suspended in a 1X binding buffer. Cells were measured by a ow cytometer (BD Biosciences).