HPLC Analysis of Intracellular Inositol Phosphate Contents.

Cells were labeled with 60 μCi [³H]-myo-inositol (PerkinElmer) in RPMI 1640 medium for 4 d. Soluble IPs were extracted from labeled cells as described (54). Inositol incorporated into lipids were measured by extracting the remaining cell pellet with 0.1 M NaOH and 0.1% Triton X-100 for 12 h at room temperature with shaking and counting a fraction of the solubilized material in a liquid scintillation counter. Soluble IP levels were normalized against total lipid inositol content. The ³H-labeled IPs were resolved by HPLC, as described previously (54).