Tlc silica gel 60 f254 20 20 cm

The pellets (5 OD units) of control and AEA (50 μg/mL, 2 h)-treated MDRSA CI-M were resuspended in 100 μL of DDW and sonicated on ice with a probe sonicator (UP200S Ultrasonic homogenizer, Hielscher Ultrasonics, Teltow, Germany) for 1 min with a 30% amplitude and 0.5 s intervals. The lipids from the sonicated samples were extracted according to the Bligh and Dyer method [135 ], and loaded onto a TLC plate (TLC Silica gel 60 F254 20 × 20 cm, Merck, Darmstadt, Germany). As controls, 5 μg of cardiolipin (CL) (Avanti Polar Lipids, Alabaster, AL, USA) and 5 μg of phosphatidylethanolamine (PE) (Sigma, St. Louis, MO, USA) were loaded on the TLC plates. For the first dimension, the plates were run in chloroform-methanol-water solution (65:25:4, v/v/v), and for the second dimension, they were run in chloroform-methanol-acetic acid-water solution (90:15:10:3.5, v/v/v/v). Then, the plates were dried for 5 min and sprayed with Molybdenum Blue reagent (Sigma, St. Louis, MO, USA). Five minutes after spraying, the images of the developed TLC plates were captured using the Fusion Solo S Imager (Vilber, Marne-la-Vallée Cedex 3, France) and the Fusion.CAPT software.

TLC of the LP extract and its fractions was performed on aluminum TLC plates (TLC silica gel 60 F₂₅₄ 20 × 20 cm, Merck, Darmstadt, Germany) using the solvent system CHCl₃/MeOH/H₂O (70:26:4, v/v/v) as the eluent. The TLC chromatograms were initially observed with UV light (254 and 366 nm) using a CAMAG TLC Visualizer and processed with the software visionCATS and winCATS. Additionally, the plates were treated with the chemical reagent ninhydrin, which is widely used for the detection of amino groups in analyzed samples. Ninhydrin reacts with primary amino groups to form purple-colored products (Ruhemann’s purple) [65 (link)]. The preparation of the reagent was performed by adding 0.2 g of ninhydrin to 100 mL of EtOH (0.2% w/v). The solution was sprayed onto the plates, followed by heating at 100 °C until the appearance of spots.