Easy Detection of PLD Activity but with Extreme Variations Between Replicates

In this assay, PLD hydrolyses phosphatidylcholine to choline, which is determined using choline oxidase resulting in a colorimetric/fluorometric product, proportional to the PLD activity in the sample.

Autotaxin belongs to the ENPP family of enzymes and displays phospholipase D (PLD) activity. For our study's purposes, BV-2 cells were seeded onto 6-well plates at a density of 1x10⁵ cells/well and serum-starved overnight prior to experiment. Then, cells were treated with LPS (20 ng/mL) in absence or presence of the indicated concentrations of PF8380 (ATX inhibitor) for the indicated time periods. DMSO was used as vehicle control. At the end of the time points, the medium was collected. 10 µL of the supernatant was mixed with 90 µL of the master reaction. After 10 min, initial absorbance, (A570) initial, was measured at 570 nm. The plate was incubated at room temperature for an additional 20 min and absorbance was measured again to determine (A570) final.

The kit is easy and fast to use. Samples should be assayed fresh! Extreme variations were observed between experiments and between replicates during the same experiment. The experiment needed to be performed at least 4 times in order to gain some confidence on the results.