A single-cell assay was developed to measure the activation of phosphoinositide 3-kinase (PI3K) using microanalytical chemical separations and a fluorescently labeled lipid substrate. Phosphatidyl-inositol 4,5 bisphosphate labeled on its acyl chain with Bodipy fluorescein (BodipyFlPIP2) was utilized as a substrate for both in vitro and cell-based assays. Detection limits for the substrate and product of the PI3K reaction were 10 to 20 zeptomol. In vitro assays with PI3K with and without pharmacologic inhibitors demonstrated that BodipyFlPIP2 was converted to phosphatidyl-inositol 3,4,5 trisphosphate (BodipyFlPIP3). BodipyFlPIP3 could be back converted to BodipyFlPIP2 by the phosphatasePTEN. When BodipyFlPIP2 was added to a cell lysate, 1.4 fmol of the BodipyFlPIP3 were produced per ng of protein in the cytoplasmic extract in 10 min. Addition of BodipyFlPIP3 to a cell lysate yielded 3 fmol of BodipyFlPIP2 per ng of protein in 8 min. Both BodipyFlPIP2 and BodipyFlPIP3 were measureable in single cells and the two species could be inter-converted. Under the appropriate conditions, a fluorescent diacylglycerol was also detected in single cells. When the FcεR1receptor on the cells loaded with the fluorescent lipid was cross-linked, the amount of BodipyFlPIP3 generated per cell increased 4-fold over that of unstimulated cells. This production of BodipyFlPIP3 was blocked by wortmannin. Chemical cytometry utilizing the fluorescent lipids will be of value in understanding lipid metabolism at the single-cell level.
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