Fluorescein isothiocyanate (fitc)

After cell seeding, the cells were pre-treated with PCS extract, UVB irradiated, and placed in medium for 24 h. Then, cells were fixed with methanol and incubated with anti-γ-H2AX serine139 (1:250) (Merck Corp., Germany) at 4 °C overnight. After that, cells were incubated with secondary antibody conjugated with FITC (1:500) (Cell Signaling, MA) and counterstained with Hoechst33342, and observed under the fluorescence microscope (DP73+IX71 Olympus, Japan). The relative green-fluorescence intensity was quantified by using Image J densitometer.

HNSCC cells were harvested from the glass coverslips and were fixed in 4% paraformaldehyde. After sealing with 10% normal goat serum for 1 h, the primary antibodies were applied and incubated overnight at 4°C. Secondary antibodies conjugated with rhodamine or FITC (Cell Signalling Technology (CST), USA, dilution 1:200) were incubated on the next day and Vectashield mounting medium containing 4′,6‐diamidino‐2‐phenylindole (DAPI) (Sigma‐Aldrich, USA) was then applied. To detect the expression and localization of p65, Triton X‐100 (Sigma‐Aldrich) was applied before sealing with goat serum. The primary antibodies used were CXCR1 (ab313462, abcam, dilution 1:50) and p65 (#8242, CST, dilution 1:400).
To visualize the phagocytosing process of sEVs, we used PKH26 to stain HN6 cells and Carboxyfluorescein N‐succinimidyl ester (CFSE, No. E607337, Sangon Biotech) to stain sEVs according to the manufacturer's instructions. CFSE‐labelled sEVs and PKH26‐labelled HN6 cells were co‐cultured for 4 h to observe the fluorescence status. Immunofluorescence and fluorescence images were captured using a fluorescence microscope (Olympus, Japan).