High-Performance Thin Layer Chromatography: A Technique for Preliminary Separation and Quantification of Lipids From Neutrophils

1. High-performance thin layer chromatography (HPTLC) to semi-quantify the amount of several lipids, including cholesterol, phospholipids, and sphingomyelin

1. Prepare the following four running solutions and staining solutions.
   1. Solution 1: Mix ethyl acetate (26.6%), 1-propanol (26.6%), chloroform (26.6%), methanol (26.6%), and potassium chloride (9.6%). Prepare KCl by dissolving 0.25 g of KCl in 100 mL of HPLC-quality water.
   2. Solution 2: Mix n-hexane (73%), diethyl ether (23%), and citric acid (2%).
   3. Solution 3: 100% n-hexane.
   4. Prepare the staining solution by mixing distilled water (90 mL) with 7.5 g of copper sulfate, and then add 10 mL of phosphoric acid.
2. Fill up to 5 mm of each solution in a separate glass chamber. Add any kind of filter paper to increase the running speed in each chamber.
3. Pre-incubate a 20 x 10 cm HPTLC silica gel 60 glass plate in the first running solution until the running solution reaches the top of the plate. Then dry it for 10 min at 110 °C.      
   NOTE: These plates can be stored for future use in aluminum foil and dried for 10 min at 110 °C prior to use.
4. Dissolve a lipid pellet obtained from human peripheral blood-derived neutrophils in 200 µL of chloroform/methanol (1:1) solution and incubate for 15 min at 37 °C to dissolve.
5. Use a ruler and a soft pencil to mark the loading spots for the desired number of samples, plus at least one standard. Mark the running distance at approximately 4 cm and 6 cm for the first and second running solution, respectively.
6. To load the samples, wash the 10 µL syringe 3 times in chloroform/methanol (1:1) prior to loading each new sample. Load 10 µL of each sample drop-wise, trying to concentrate the sample on as small an area as possible. Samples should be loaded at least in duplicates.
7. Place the plate vertically into the first chamber with running solution 1 (step 1.1.1). Ensure that the plate is parallel to the wall of the glass chamber to achieve a uniform migration speed.
8. Once the solvent line has reached the first mark, remove the plate, dry it, and place it in the second solution. Repeat similar steps for the second and for the third running solutions; leave the plate in the solution until the solvent front reaches the top of the plate, then remove and dry it at RT for 1 min.
9. Place plate in copper sulfate solution for 7 s. Remove the plate, dry it thoroughly, and bake it in an oven for 7 min at 170 °C. Wait for the plate to cool.
10. Using a thin layer chromatography and gel analysis software, as well as an image processing software, scan and analyze to identify the resolved bands of lipids.