Fig. 3.

ATP-induced 2-AG production requires sustained rise in [Ca²⁺]_i. (a and b) Microglial cells were preincubated for 30 min with vehicle (control), EGTA (1 mM), or BAPTA-AM (10 μM) and incubated for 10 min with ATP (1 mM) or Bz-ATP (200 μM). Lipids were extracted and HPLC-purified, and amount of eCB was quantified by chemical ionization-GC/MS. Values are mean ± SEM of n = 6-62 independent eCB quantifications, each performed on one 60-mm dish (i.e., 3-31 separate experiments performed in duplicate). ^**, P < 0.01, significantly different from basal (dotted line) (ANOVA followed by Dunnett's post test). (c and d) [Ca²⁺]_i in microglial cells was determined as described in Materials and Methods. Ratio plots in figures were averaged from ≈25 cells in the field of view and are representative of three experiments.