Isolation and identification of exosomes secreted from HK-2 cells exposed to high oxalate

Abstract

Objective To investigate the method for isolating and identification exosomes from the HK-2 cells exposed to high oxalate. Methods HK-2 cells were cultured to serum-free culture medium and treated with oxalate at the concentration from 0 mmol/L to 10.00 mmol/L for 48 h. CCK-8 assays were performed to measure the cells proliferation.Combined the cell morphology, observed under inverted microscope with statistical analysis, we finally 2.00 mmol/L oxalic acid as the experimental concentration. The HK-2 cell was exposed to 2.00 mmol/L oxalate for 48 h. The supernatants were collected. Exosomes were isolated and purified from the supernatants by ultracentrifugation. Transmission electron microscopy (TEM) was used to observe the morphology of isolated exosomes. Particle size of exosomes were detected with Nanosight technology. Western blot analysis was used to examine the experession of HSP70, CD63. Results CCK-8 assay showed that the cell viability in each group, including (100.0±4.0)% in 0 mmol/L group ; (97.7±1.5)% in the 0.25 mmol/L group; (97.3±2.1)% in the 0.50 mmol/L group; (87.7±2.1)% in the 1.00 mmol/L group; (76.0±1.0)% in the 2.00 mmol/L group; (58.1±2.6)% in the 4.00 mmol/L group; (52.7±1.5)% in the 5.00 mmol/L group; (37.7±3.2)% in the 8.00 mmol/L group ; (31.3±2.0)% in the 10.00 mmol/L group.The isolated exosomes demonstrated round or oval shape under TEM. The peak particle size was 56 nm, which the overall mean particle size was 87 nm. Those and particles with a diameter between 30-150 nm accounted for 91.2%. In this experiment, The expression of HSP70, CD63 could be detected in the isolated exosomes.However, only the expression of HSP70 could be detected in the HK-2 cells. Conclusions Under the treatment of 2 mmol/l oxalate for 48 hours, Ultracentrifugation can be used to isolate and purify exosomes efficiently from the HK-2 cells. This is helpful for further study of exosome as mediator of cell-to-cell communication. Key words: Exosomes; Kidney stone; Oxalate.