Quantification of Inflammatory Markers

The cell culture was collected, and levels of TNF-α (#SRP3177), IL-8 (#SRP3311), MCP-1 (#SRP3109), ICAM-1(#SRP6491), and VCAM-1(#RAB0505) were measured using correlating ELISA kits (Sigma-Aldrich, USA). Briefly, the standards were diluted to 5 gradient concentrations and were added to a 96-well plate along with the supernatants collected from each cultural medium. After incubation for 30 minutes at 37°C, the medium was removed, and the wells were washed using the washing solution. Subsequently, the wells were added with conjugate reagents followed by incubation for 30 minutes at 37°C. After washing, the TMB solution was added for coloration at 37°C for 15 minutes, followed by adding the stop solution to terminate the reaction. Lastly, the absorbance at 450 nm was measured using the microplate reader (Mindray, Shenzhen, China) [31 (link)].