PLA2 biochemical activity assays

Cytosolic and secreted PLA₂ activity of recombinant PLA2G4A, heat-killed S. aureus preparation and ILC2 culture supernatant were measured using either a cytosolic PLA₂ kit (Cayman Chemicals) or a secretory PLA₂ kit (Cayman Chemicals) according to the manufacturer’s protocols. Arachidonoyl thio-PC is the substrate for cPLA₂ and hydrolysis of the arachidonoyl thioester bond at the sn-2 position by cPLA₂ releases a free thiol which can be detected by DTNB (5,5′-dithiobis[2-nitrobenzoic acid]) producing a colored precipitate. For the sPLA₂ kit in the presence of PLA2, cleavage of the substrate (diheptanoyl thio-PC) at the sn-2 position results in release of the thiol group. In both assays absorbance was measured with a spectrophotometer (Clariostar) (wavelength 414 nm) to give a measure of PLA2 activity.