Cytokine analysis using ELISA.

Cell culture medium was collected 24 h post-antibiotics and centrifuged (2,400 relative centrifugal force [rcf] for 10 min) to remove bacterial and cellular debris; supernatants were stored at −20°C. Samples were thawed on ice and centrifuged at 16,000 rcf for 5 min prior to cytokine analysis. The following kits were used: IL-1β human ELISA kit, EH2IL1B2 (Thermo Fisher Scientific); MIG human ELISA kit, EHCXCL9 (Thermo Fisher Scientific); IL-6 human ELISA kit, KHC0061 (Thermo Fisher Scientific); MCP-2 human ELISA kit, EHCCL8 (Thermo Fisher Scientific); RANTES human ELISA kit, EHRNTS (Thermo Fisher Scientific); and IL-10 human ELISA kit, ab100549 (Abcam). MIG is often referred to as CXCL9 (C-X-C motif chemokine ligand or monokine induced by gamma interferon), and MCP-2 and RANTES are referred to as CCL8 (C-C motif chemokine 8) and CCL5 (C-C motif chemokine ligand 5). The average cytokine concentrations (pg/ml) were quantified per condition, and the standard deviation of the mean was calculated using pooled data from at least 3 independent biological replicates.