Fig. 1
Comparable responses of Q7/7 and Q140/140 microglia stimulated with LPS. A Microglia were stimulated with LPS (100 ng/ml) for the indicated times. Expression of Il-1b, Il-6 and Tnf mRNA at each time-point was normalized over the geometric mean of three housekeeping genes (Normalization Index). N ≥ 3. B TNF secreted in the conditioned medium was estimated by ELISA. N ≥ 3. C Nitrite in the conditioned medium. N = 8 for naïve microglia, N ≥ 4 for stimulated microglia. No statistically significant differences between genotypes were found for any of the measurements indicated above at any of the time points, except for TNF secretion at 6 h of LPS stimulation. D Proteins secreted in the conditioned medium after 9 h of cell treatment with 100 ng/ml LPS were quantified using a Luminex® multiplex assay. No statistically significant differences between genotypes were found for any of the cytokines and proteins measured. N = 5. E, F Dose–response of LPS stimulation. Microglia were stimulated for 6 h with LPS at the indicated concentrations and mRNA was quantified by qPCR. No differences in the expression of Il-1b, Il-6 and Tnf mRNA (E), and the amount of TNF released in the medium (F) were observed between Q7/7 and Q140/140 microglia at all concentrations of LPS tested. N = 3 for Q7/7 and N = 4 for Q140/140 microglia. G Microglia were primed with GM-CSF for 4 days, followed by 1 h incubation in IFN-γ and 48 h in LPS to induce a pro-inflammatory state. Expression of Tnf, Il-6 and Il-10 was normalized as in (A). N ≥ 4. H mRNA expression of Spi-1 was measured in control conditions and after stimulation for 12 h with LPS (100 ng/ml) and normalized as in (A). N ≥ 4. In A–C and E–F, a one-way ANOVA was used to confirm the effect of treatment in each genotype. The paired two-tailed t-test was used to compare differences between genotypes at each time point and LPS concentration. In D, two-tailed t-test was used to compare cytokine expression between genotypes. In H, a two-way ANOVA with Tukey’s comparisons test was used. Bars are mean values ± STDEV. *, p < 0.05; **, p < 0.01