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. 2017 Sep 22;7:12158. doi: 10.1038/s41598-017-12529-7

Figure 3.

Figure 3

Addition of microglia to L-leucyl-L-leucine methyl ester (L-LME)-treated astrocytes fails to fully restore TNF-α and LPS-induced increases in Saa1 mRNA. Enriched cortical astrocytes (250,000 per 24-well) were treated with 50 mM L-LME for 60 min, and returned to fresh culture medium for 24 h. After this time (medium change to DMEM + 0.5% FCS) purified cortical microglia were added, at the numbers indicated on the horizontal axis (+5 K, + 10 K, + 20 K, + 250 K) to the astrocyte cultures and incubation continued for a further 24 h. Cultures were then challenged with either 10 ng/ml TNF-α or 100 ng/ml LPS and cells processed for Saa1 mRNA expression by qRT-PCR 6 h (a) and 24 h (b) later. Data are presented as relative expression level (normalized with respect to β-actin (βACT)) at each time point and are means ± s.e.m. n = 3. *p < 0.05 vs control (CTRL); **p < 0.01 vs control; ***p < 0.001 vs control. ##p < 0.01 vs TNF-α; ###p < 0.001 vs TNF-α. Similar results were obtained in a second experiment. (c) The same numbers of microglia were cultured in a parallel 24-well plate and subjected to the same treatments as above and then processed for Saa1 mRNA expression by qRT-PCR at 24 h. For comparison, samples plated with 250,000 microglia per well were also analyzed. Data are presented as relative expression level (normalized with respect to β-actin (βACT)) and are means ± s.e.m. n = 3. ***p < 0.001 vs all other cell densities; ###p < 0.001 vs TNF-α.