Fig. 2: Contactless co-cultures of neurons and stimulated microglia increases the neuronal expression of REST.
a, b Timeline (a) and schematics (b) of the experimental procedures for the contactless microglial/neuronal co-cultures. Primary microglial cells were harvested from the mixed primary glial cultures at 18 DIV and seeded on matrigel-coated TranswellsTM for 1 day before being treated for 24 h with either LPS (Stim) or vehicle (NS). After treatment, microglia-coated transwells were added to 14 DIV primary cortical neurons that were harvested 24 h later. c Microglial activation was verified by staining with the microglial marker Iba1. DAPI-stained nuclei are shown in blue. Scale bar, 25 µm. d After 24 h of contactless co-culture with either control or activated microglia, TranswellsTM were removed and neurons were harvested and subjected to qRT-PCR to assess the full-length REST expression. Gapdh, Actin, and Hprt1 were used as housekeeping genes. Bars show mean ± sem with superimposed individual points obtained from three independent preparations. Similar to activated T cell supernatant, the secretory activity of activated microglia increases REST expression in the co-cultured neurons. *p < 0.05; Mann–Whitney U test.
