Defined aliquots of meibum or standard lipids were evaporated to dryness and incubated with 120 μl of 95 % sulfuric acid at 95 °C for 20 min in individual glass tubes. Then, the reaction mixtures were rapidly cooled by placing the tube rack on an ice pack, the tubes contents were vortexed and a 100-μl aliquot transferred to individual wells of a 96-well glass plate (Zinsser Analytic GmbH, Germany; purchased through Aldrich, Milwaukee, WI, USA; catalog number Z40644-9). Initial pre-vanillin absorbance was measured using a Beckman Coulter DTX880 Multimode Detector (Beckman Coulter, Indianapolis, IN, USA) and a 535-nm filter. This was followed by addition of 50 μl of 0.2 mg/ml vanillin in 17 % aqueous phosphoric acid, incubation at room temperature for 10 min in the dark and recording of a post-vanillin chromophore absorbance at the same wavelength (A535). The final SPV response of samples was defined as the difference between the final post-vanillin and pre-vanillin 535 nm absorbance readings. In assays examining the effects of proteins or mucins, these components were added to the dry lipid samples before addition of sulfuric acid.