Table 2.

Recommended applications and detection methods for IMPACT and RT-IMPACT.

Detection method	IMPACT	RT-IMPACT
Flow cytometry (analytical)	Profiling of PLD activity within and across different cell lines under various stimulants	Similar to IMPACT
Fluorescence-activated cell sorting (preparative)	Enrichment of cells with high or low PLD activity within a population; availability and stability of reagents make this a well-suited application	Not recommended for this application, due to high value and limited aqueous stability of reagents
Fluorescence microscopy	Overall cellular fluorescence reflects PLD activity; however, observed subcellular localizations of IMPACT-derived fluorescence may not reflect accurate localizations of PLD activity due to fluorescent lipid trafficking	Rapid, real-time imaging protocol can reveal both extent and accurate subcellular localizations of PLD activities, with minimal interference from lipid-trafficking events due to the rapid timescale of the RT-IMPACT protocol. N.B.: Extended time-lapse movies of RT-IMPACT may be used to visualize intracellular trafficking of fluorescent lipids to study lipid transport mechanisms
Fluorescence-coupled HPLC	Determination of PLD activity at the bulk population level; availability and stability of reagents make this a well-suited application	Not recommended for this application, due to high value and limited aqueous stability of reagents
Mass spectrometry	Determination of precise lipid species (defined by acyl tail composition) produced by active PLDs, reflecting substrate preferences	Similar to IMPACT but with lower signal-to-noise ratio