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Figure 5 | Journal of Neuroinflammation

Figure 5

From: Interferon regulatory factor 8/interferon consensus sequence binding protein is a critical transcription factor for the physiological phenotype of microglia

Figure 5

Purified Irf8 +/+ and Irf8 -/- microglia in vitro. (A-D) Representative phase contrast (A, B) and immunocytochemical (C, D) pictures of the microglia isolated from the Irf8 +/+ (A, C) and Irf8 -/- (B, D) mixed glial cultures grown in the presence of M-CSF (20 ng/ml) by magnetic-activated cell sorting. Purified cells were immunolabeled for CD11b (green in C, D) at 24 h after isolation. Nuclei were counterstained with DAPI (blue). Scale Bar: 50 μm. (E) IRF8 and PU.1/SFPI1 mRNA levels in the purified Irf8 +/+ microglia. Purified microglia were incubated in medium alone (control) or in the presence of IFNγ (IFNG, 100 ng/ml), lipopolysaccharide (LPS, 100 ng/ml) or both for 24 h. Reverse transcribed IRF8 and PU.1/SFPI1 cDNA levels are plotted as ratios to copy numbers of β-actin cDNA on a logarithmic scale. (F) Immunoblots for IRF8 confirmed the results of mRNA. (G) Purified Irf8 -/- CD11b+ microglia were less proliferative than Irf8 +/+ CD11b+ microglia even in the presence of exogenous M-CSF. Purified microglia were preincubated with medium alone for 24 h after isolation from Irf8 +/+ (open bars) and Irf8 -/- (closed bars) mixed glial cultures, and then treated with medium alone (control), or medium supplemented with M-CSF (20 ng/ml) or GM-CSF (20 ng/ml) for 24 h. After a 6 hour incubation with EdU, EdU-positive and DAPI-positive nuclei were counted. **P < 0.01 in comparison with control or between the two groups indicated.

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