Quantification of phospholipids content by phosphorus assay

The total amount of phospholipids in each lipid extract was determined in two replicate experiments by Barlett and Lewis colourimetric assay, based on the measurement of inorganic phosphorus, as done in our laboratory^23,24. Briefly, an aliquot of 10 µL of the extract was dried and incubated 1 h at 180 °C with 125 µL of perchloric acid (70%) in a heating block (Stuart, U.K.) to hydrolyse the inorganic phosphorus of the phospholipids. Afterwards, and once cooled down to room temperature, the solutions were mixed by vortexing with 825 µL of Milli-Q water, 125 µL of (NH₄)₆Mo₇O_24·4H₂O) 2.5% and 125 µL of ascorbic acid 10% freshly prepared. Samples and standards (8 standards solutions, with different concentrations of phosphorus) were simultaneously incubated at 100 °C in a water bath for 10 min and cooled down. The content of inorganic acid was measured in a microplate spectrophotometer (Multiscan 90, ThermoScientific) at 797 nm.