Hptlc silica gel aluminum back plate

The study of radiolabeled inositol incorporation was based on procedures described previously with substantial modification[26 (link)-28 (link)]. T. forsythia was grown in the presence of 1 μCi of [¹⁴C(U)] myo-inositol (specific radioactivity 200–250 mCi/mmoL) as described in Section 2.1. After 4 days of growth, the biomass was pelleted and lipids were extracted using the Bligh–Dyer method [22 (link)]. 80 μL of water were added to the wet biomass followed by 300 μL of chloroform:methanol (1:2), vortexed for 5 min and left shaking for an additional 10 min. Subsequently, 100 μL of chloroform were added followed by 100 μL of water, vortexing for 5 min and left shaking for 10 min, after each addition. After phase separation, the top aqueous phase was carefully removed. The bottom organic phase was then backwashed three times by addition of a 100 μL of water. The radioactivity in each wash fraction and in the organic phase was determined using a liquid scintillation analyzer Tri-Carb 2910 TR (Perkin Elmer, Waltham, MA, USA). The organic phase containing the extracted lipids was dried under a flow of nitrogen, redissolved in chloroform:methanol (8:2), spotted onto a HPTLC silica-gel aluminum-back plate (Merck) and then chromatographed in solvent A. The plate was then subjected to autoradiography using Kodak BioMax XAR film (Sigma-Aldrich) for three weeks at −80 °C.