Ab190805

h‐JBMMSCs were cultured in osteoblast‐inducing conditional medium in 24‐well plates with or without APN. The cells in the APN‐treatment groups were cultured for 7 days with the addition of APN three times (once every 3 days). All the cells and supernatants were collected for ELISA. Total protein was extracted using the RIPA buffer (Applygen, Beijing, China), and the protein concentration was determined using the bicinchoninic acid reagent (Thermo Fisher Scientific, New York, NY, USA). ELISA was then used to assess the levels of CXCL1 (ab190805; Abcam, Cambridge, United Kingdom) and CXCL8 (ab174442; Abcam, Cambridge, United Kingdom) in the cytoplasm and supernatant according to the manufacturers’ instructions.

The production of chemokine ligands CXCL1, CXCL2, and CXLC8 by melanoma cells and primary melanocytes was determined by ELISA of cell culture supernatants corresponding to the same cell culture and sEV isolation conditions described above (Methods Section 4.1, Section 4.2). Abcam human ELISA kits ab190805, ab184862, and ab214030 were used according to manufacturer’s instructions to detect cellular production of CXCL1, CXCL2, and CXCL8, respectively.

CXCL1 and CXCL3 were determined in cell supernatant, serum, and tumor tissues. Cell supernatant was collected and filtered by 0.45 mm strainer. Tumour tissues was weighed, grinded, and treated with Cell Extraction Buffer PTR (Abcam) containing a protease inhibitor cocktail (Roche, Switzerland). The serum was separated from blood of tumor bearing mice. Mouse CXCL1 (ab216951, Abcam), mouse CXCL3 (ab272191, Abcam), human CXCL1 (ab190805, Abcam) or human CXCL3 (ab234574, Abcam) ELISA kits were performed according to the manufacturer’s instructions.