Hptlc plate

Alcian blue, aluminium chloride, Folin-Ciocalteu reagent, ethylene diamine tetra acetic acid (EDTA), 5,5-dithio-bis-(2-nitrobenzoic acid (DTNB), gallic acid, thiobarbituric acid, trichloroacetic acid, toluene, ethyl acetate, formic acid, formaldehyde, sodium hydroxide, rutin, phosphoric acid, pyrogallol, haematoxylin, eosin were purchased from HiMedia Laboratories Pvt Ltd, Mumbai, India, and HPTLC plates, tris-HCl buffer, ethanol, amentoflavone were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA).

Pyrex glass tubes (30x100mm & 18x100mm)
Acid-washed glass beads (0.4 - 0.6 mm, VWR, LENZ05124005)
HPTLC plates (Sigma/Merck 1.05641.0001)

To analyse lipid content of liposomes and EVs by thin-layer chromatography, total lipid was isolated using the Folch method. For this, 25 μl sample was mixed with 500 μl solvent consisting of a 2:1 (v/v) ratio of chloroform and methanol, followed by 5 min of sonication in a bath sonicator and 2,000 rpm shaking for 20 min at 4 °C. Then 125 μl water was added to induce phase separation, and samples were centrifuged at 2,000g for 5 min. The upper aqueous layer was removed, and the lower phase was transferred to new vials. Solvent was then evaporated under nitrogen, and 25 μl chloroform was added before shaking for 5 min at 2,000 rpm and sonicating for 5 min. High-performance thin-layer chromatography (HPTLC) plates (Sigma-Aldrich) were activated by heating to 110 °C for 30 min, and samples were then spotted in 3 μl volume using graduated glass pipettes (Hirschmann Ringcaps). The running chamber was filled with mobile phase, consisting of methyl acetate, isopropanol, chloroform, methanol and aqueous KCl (0.25) at a ratio of 25:25:25:10:9, as described previously^{83 (link)}. Plates were then inserted and allowed to run until the solvent front had reached within 0.5 cm of the upper plate limit. Plates were then air dried for 10 min and developed in an iodine vapour chamber. After 30 min, plates were immediately imaged in a GelDoc (Bio-Rad).