Phospholipid assay kit

Mouse tissues (brain, retina and RPE/choroid/sclera) were homogenized with a lipid extraction buffer: Hexane: isopropanol (3:2) and incubated for 30 min at room temperature. The organic solvents were centrifuged at 1500× g for 10 min and the supernatant was collected and dried at 50 °C. The dried solvent was dissolved with 1× reaction buffer. Total cellular cholesterol and phospholipid were measured using an Amplex^® Red Cholesterol Assay Kit (Alfa Aesar, Heysham, UK) and a Phospholipid Assay Kit (Sigma, Dorset, UK), respectively, according to the manufacturer’s guidance. For measurement of triglyceride, mouse tissues were homogenized in buffer containing 5 volumes of isopropanol, 2 volumes of water and 2 volumes of Triton X-100 and centrifuged for 4 min at 13,000 rpm. The supernatants were collected and subjected to triglyceride measurement using an EnzyChrom Triglyceride assay kit (BioAssay system, Hayward, CA, USA), according to the manufacturer’s protocol.

The BESC and HESC were grown at a density of 100,000 cells/well in 12-well tissue culture plates and treated with siRNA or inhibitors, as described above. After the treatment period, the cells were collected in 200 µl/well 1X cholesterol assay buffer and stored in Eppendorf tubes at −20°C. When needed, the samples were defrosted at room temperature and sonicated for 10 min in a sonicating water bath. Cellular cholesterol content was measured using the Amplex® Red Cholesterol Assay Kit (Invivogen), according to the manufacturer’s guidelines. Total cellular phospholipid was measured in the samples prepared for the cholesterol assay using a phospholipid assay kit (Sigma Aldrich, MAK122), according to the manufacturer’s guidelines. The cholesterol concentrations were then normalized to the phospholipid concentrations, and the data are expressed as arbitrary units.

The effect of EFL₃ on phospholipid content was measured following the methods reported before [22 (link)]. Mycelia were cultured as described above in Section 2.6. After being collected and washed with sterilized distilled water, mycelia samples were ground with liquid nitrogen. Subsequently, 10 mg mycelia per sample were collected and resuspended in the assay buffer. The phospholipid content of P. capsici mycelia were measured using a phospholipid assay kit (Sigma-Aldrich, St. Louis, MO, USA). The effect of EFL₃ on cellulose content was also determined. Mycelia were cultured and collected as described above. After being ground with liquid nitrogen, 0.3 g of mycelia per sample were collected and resuspended in the extraction buffer. The cellulose content of P. capsici mycelia were measured by a cellulose assay kit (Solarbio, Beijing, China). The experiment was repeated three times.

In order to investigate the release of liposome under different pH, 71.6 g disodium hydrogen phosphate (Na₂HPO₄) and 31.2 g sodium dihydrogen phosphate (NaH₂PO₄) were dissolved in 1000 ml deionized water, respectively, and configured to obtain 0.2 M Na₂HPO₄ and NaH₂PO₄ mother liquor, and PBS solutions could be obtained via mixing the mother liquor according to Supplementary Table 3.
MSaP-aL/p was immersed in 50 ml centrifuge tubes containing 10 ml pH 7.4, pH 6.6, and pH 5.8 PBS, respectively. All centrifugal tubes were placed at 37 °C, on the 120 cycles min⁻¹ constant temperature vibrator (Thermo, USA). After 3, 6, 9, 12, 15, 18, 21, and 24 h of vibration, the resulting solution was collected and stored at −20 °C with 10 ml fresh PBS re-added into the tubes. The effects of different environment pH on Schiff base breakage and liposome release was studied using the Phospholipid Assay Kit (Sigma-Aldrich, MAK122, USA). Similar to the above methods, the effects of different acidic environments on the release of NGF from microsol electrospun fiber scaffolds were also analyzed using the NGF ELISA Kit (R&D Systems, USA). The cumulative release curves were drawn, respectively.