Fig. 2:

Lipid synthesis and DAG production in small numbers of primary B cells. (A): Kinetics of lipid synthesis in primary B cells. Incorporation of [³H]palmitic acid (2.5 mCi/ml) into lipids was determined in purified wt primary B cells. Lipid extraction was performed according the protocol of Folch (10). Lipid synthesis is expressed in cpm and represents the average + SEM of three independent performed in triplicate. (B): BCR-induced DAG production is dose dependent. 5x10⁶ purified B cells were labeled for 6h with [³H]palmitic acid (2.5 mCi/ml) and stimulated for 5 min with various doses of anti-IgM. Lipids were extracted and separated as described (12) and DAG was quantified. DAG is expressed as percentage increase of DAG of total radioactivity and represents the average + SEM of three independent performed in triplicate. *P<0.05; ***P<0.001. (C): [³H]palmitic acid is incorporated into whole lipids in small numbers of primary B cells. The indicated numbers of non-stimulated B cells from wt mice were labeled for 6h with [³H]palmitic acid (2.5 mCi/ml) and total incorporation of [³H]palmitic acid into lipids (r=0.998) was monitored as described (6). (D): BCR-induced DAG production can be measured in as few as 0.5x10⁶ primary B cells. The same protocol as in (B) was applied to 0.5-5x10⁶ primary B cells that were stimulated for 5 minutes with 20 μg/ml of anti-IgM. ***P<0.001