6% FBS is determined to be the optimal FBS % in growth medium to activate macrophages.

<p>(<b>A</b>) U-937 macrophage cells were activated for 24 h using 66.7 μg/mL or 200 μg/mL of FBS-free eGFP medium or FBS-free wild-type hCLCA1 medium with 3%, 6%, or 10% FBS growth medium. IL-8, IL-6, IL-1β, TNF-α, IL-12a and IL-10 were quantified by their mRNA expression using RT-qPCR. The fold difference at each concentration was compared against eGFP (the control) at the same concentration. Results were the means of 5 samples ± SEM. Each sample was a result of an individual transfection paired with an eGFP transfection. Significant fold differences from corresponding control values (eGFP of the same concentration) are indicated by * (<i>p</i> < 0.05), ** (<i>p</i> < 0.005) or *** (<i>p</i> < 0.001). (<b>B</b>) Representative western blot analysis probing against hCLCA1 N-terminal antibody was utilized in conjunction with Coomassie gel staining to compare the total protein contents between FBS-containing hCLCA1 medium and FBS-free hCLCA1 medium. Both western blot and coomassie gel staining showed that hCLCA1 was one of the major secreted molecules, and there was less impurities in the FBS-free hCLCA1 medium without a major loss in hCLCA1 protein.