Silica gel 60a plates

Total lipid extracts were analyzed by TLC on silica gel 60A plates (Merck, 20 × 10 cm, layer thickness, 0.2 mm). The plates were washed two times with chloroform/methanol (1:1, by vol.) and activated at 180°C before use. Polar lipids were eluted with an acid solvent (chloroform/methanol/acetic acid/water, 85:15:10:3.5, by vol.). Total lipid detection was carried out by molybdenum blue spray reagent (Sigma-Aldrich) specific for phospholipids (Kates, 1986 ). Alternatively, total lipid detection was done with reversible staining exposing the TLC plate to iodine vapor for 4–5 min for staining all classes of lipids before the lipid bands isolation. To analyze in detail the various components of the lipid extracts, each band present on the plates was scraped and lipids extracted from silica, as previously described (Kates, 1986 ); briefly, 0.5 ml of a mixture chloroform/methanol/water (1:2:0.8, by vol.) has been added to silica bands, and the samples were vigorously stirred and centrifuged. Lipid bands of preparative TLC were analyzed by positive and negative ion mode MALDI-TOF/MS.

Total lipid extracts were analyzed by thin-layer chromatography (TLC) on silica gel 60A plates (Merck, 20 × 10 cm, layer thickness 0.2 mm). The plates were washed twice with chloroform/methanol (1:1, by volume) and activated at 180 °C before use. Polar lipids were eluted with Solvent A (chloroform/methanol/acetic acid/water 85:15:10:3.5, by volume); the neutral lipids were separated by TLC in Solvent B (hexane/diethyl ether/acetic acid, 70:30:1, by volume).
Lipid detection was carried out by spraying the plate with 5% sulfuric acid in the water, followed by charring at 180 °C for 5 min, or exposing the TLC plate to iodine vapor, for staining all classes of lipids. The estimation of the content of individual polar and neutral lipids of the total lipid extracts was performed by video densitometry analysis of spots on TLC, obtained after averaging three replicates of C, NI, and NII (ImageJ software).

Total lipid extracts were analyzed by TLC on silica gel 60A plates (Merck, 20 × 10 cm, layer thickness 0.2 mm). The plates were washed twice with chloroform/methanol (1:1, by volume) and activated at 180°C before use. Total lipids were eluted with Solvent A (chloroform/methanol/acetic acid/water 85:15:10:3.5, by volume).
Lipid detection was carried out exposing the TLC plate to iodine vapor, for staining all classes of lipids. Moreover, the Azure A staining were performed in order to identify the sulfur-containing lipids in the TLC bands (Kates, 1986 ).
In order to analyze the various components of the lipid extracts in detail, bands present on plates were scraped and lipids extracted from silica were then analyzed by negative and positive ion modes MALDI-TOF/MS.
Briefly, the polar lipid components of the total lipid extracts of semen samples were separated by TLC in Solvent A. Lipids were visualized by staining with iodine vapor and then were eluted and recovered from the scraped silica, as previously described (Lobasso et al., 2012 (link); Lopalco et al., 2017 (link)). Isolated and purified phospholipids were dissolved in chloroform (1 mg/ml).