Figure 4. Extracellular and intracellular IL-1β content derived from enriched cortical astrocytes incubated with LPS ± ATP.

Enriched cortical astrocytes cultured in a 96 well plate were incubated in serum-free medium with 1 μg/ml LPS for 2 h followed by addition of 5 mM ATP or not. After a further 60 min incubation culture medium (a) and cell lysates (b) were collected for IL-1β analysis. In some cases cells and prior to LPS/ATP challenge, cells were treated the previous day with 50 mM Leu-Leu-OMe (L-LME) for 60 min, and returned to fresh culture medium for 24 h. Panel (c) presents the sum values for intracellular + extracellular IL-1β. Total IL-1β content for LPS and LPS/ATP did not differ significantly, indicating that the reduced intracellular content for LPS/ATP (panel B) could be accounted for by that released into the culture medium (panel A). Production of the ATP-dependent component of IL-1β was prevented by inclusion of the selective P2X₇R antagonist A-740003 (not shown). Data are means ± s.e.m. (n = 3).