Figure 1. Experimental design to study the effect of TNF-α on ARPE-19 secreted proteins. ARPE-19 cells were grown in medium where Lys and Arg were replaced by ¹⁵N, ¹³C₆-Lys, and ¹³C₆-Arg. In parallel, ARPE-19 cells were cultured in medium with normal Lys and Arg. After cells were fully labeled with heavy and light amino acids, 10 ng/ml of TNF-α was added into each flask of labeled cells and unlabeled cells and incubated for 24 h in serum-free medium while the other remaining flasks were incubated in the same serum-free medium without TNF-α. A: For the control experiment, spent medium from labeled cells (e.g., untreated) was mixed at a 1:1 ratio with spent medium from unlabeled cells (e.g., untreated). B: To check the effect of TNF-α on ARPE−19 secreted proteins, we mixed the spent medium from labeled cells (e.g., treated with TNF-α) at a 1:1 ratio with spent medium from unlabeled cells (untreated). C: To validate the data, we performed a reverse experiment in which spent medium of labeled cell (e.g., untreated) was mixed at a 1:1 ratio with unlabeled cells (e.g., treated with TNF-α). Sample pairs were then processed as described in the method for subsequent LC-MS analysis and determination of protein ratios.