2.3. Cytokine Array

The cytokine array analysis was performed according to the method of a previous study [30], with some modifications. Cells were untreated (control) or treated with chrysophanol, and the medium was replaced with serum- and antibiotic-free DMEM for the final 24 h of the incubation. Medium from three wells was pooled, clarified by centrifugation at 200× g at 4 °C, and immediately applied to the Human Cytokine Array Kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). The cytokine array signal was detected at multiple exposure times, ranging from 15 s to 10 min. The film was scanned using a ChemiDocTMXRS + System (Bio-Rad Laboratories, Hercules, CA, USA). Signal levels were measured using ImageJ software, with the Protein Array Analyzer plugin 16.

The Human Cytokine Array Kit was provided by R&D Systems and was used according to manufacturer’s introduction and the method suggested by a previous study [31]. The image of spots was scanned and measured using NIH ImageJ software with the Protein Array Analyzer plugin 16.