To investigate the applicability of both Hnrnpab and Stx5a as reference genes in differently sourced macrophages, we compared the expression levels of six of the 11 candidate genes (three of the most stable and three of the least stable) in murine peritoneal macrophages and in the RAW 264.7 murine macrophage cell line. For all genes the Ct values were recorded for control and LPS stimulated (10 ng/ml) cells at 6 h (Figs. 3a-d) and 24 h (Figs. 3e-h). Consistent with the stability of genes in LPS stimulated BMDMs, there was no difference in Ct values observed for Hnrnpab, between the control and LPS treated cohorts, at 6 h. In addition, Stx5a was again identified as the most stably expressed gene between control and treated cells 24 h after LPS treatment, with no difference seen in Ct values between the two treatments. This additional study therefore validates Hnrnpab and Stx5a as suitable reference genes in these multiple sources of macrophages and at multiple time points.Fig. 3Stability ranking of select reference genes in LPS treated RAW 264.7 cells and peritoneal macrophages at 6 h and 24 h. Ct values were recorded for control and LPS (10 ng/ml) stimulated RAW 264.7 cells and peritoneal macrophages at 6 h (a-d) and 24 h (e-h). Ct values were transformed as instructed and applied to the 3 reference gene analysis softwares. The tables above show the ranking of most to least stably expressed reference genes between control and LPS stimulated cells in (a, e). RAW264.7 cells and (b, g) peritoneal macrophages, from top to bottom, as identified by Normfinder, GeNorm and BestKeeper softwares. The Ct values are plotted for LPS stimulated (c, f). RAW 264.7 cells and (d, h) peritoneal macrophages. The error bars represent means ± SDs. The significance values were calculated by comparison of control and treated samples as a group, at each time point. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001