Cytokine Profiling via Indirect ELISA

The Indirect Enzyme-Linked-Immunosorbent-Assay (ELISA) was applied to measure inflammatory cytokines as described in previous studies 3 (link), 17 (link). In brief, the samples with the same amount of protein were loaded into the polyvinyl chloride ELISA microplate (Corning) overnight at 4 °C. After 3 washes, the blocking buffer was loaded into the microplate for another 1 h. After removing the blocking buffer, the following antibodies were added: TNF-α, NFκB, IL-1β, IL-4, IL-10, and IL-13, and incubated at room temperature for 1 h. Afterward, TMB development solution was added and incubated for 30 min. After adding the stop solution, the microplate was read at 450 nm using a spectrophotometer (Bio-Rad; Hercules, CA, USA). All data were calculated and expressed as percent changes versus the WT group.