Fig. 3. Ca²⁺ is required for STAg-induced production of IL-12. Macrophages pre-treated with normal buffer (black bars) or BAPTA-AM (15 µM) in Ca²⁺ free/EGTA (white bars) were stimulated with STAg (50 µg/ml) for 5 hours in normal media or Ca²⁺ free EGTA medium, respectively. mRNA was isolated and reverse transcribed to cDNA, which was used in real-time PCR reactions for IL-12p35 and IL-12p40. All amplifications were conducted in triplicate and results are normalized to endogenous 18S and expressed as fold change in mRNA relative to untreated controls for each condition, according to the Ct method. Experiments were repeated at least three times with similar results. The data presented are from a representative experiment. *P0.01 by Student's t-test.