Glycolipid-Induced Cell Death Patterns

To assess the distinct morphological pattern of cell death induced by Glycolipids in HaCaT and SK-MEL-28 melanoma cell line, the cells were stained with acridine orange (AO) and propidium iodide (PI) (Merck, Gillingham, UK) [31 (link)]. HaCaT and SK-MEL-28 cells were cultured, seeded onto Nunc 12-well cell culture plates and treated with glycolipid and synthetic SLES preparations as previously described in Section 2.4. Following glycolipid treatment, the cells were washed three times with PBS (ThermoFisher Scientific, Loughborough, UK) to remove floating cells and subsequently incubated with AO and PI for 3 min, each prepared at a working concentration of 100 ug mL⁻¹ and mixed at a ratio of 1:1. Cells were then rewashed three times with PBS (ThermoFisher Scientific, Loughborough, UK) and stained cells were imaged at 200× magnification using and Eclipse TS100 fluorescence microscope (Nikon Europe B. V., Amsterdam, The Netherlands). The excitation and emission wavelengths for AO were 493 and 535 nm, and for PI they were 535 and 614 nm. Three images per well were randomly selected (by computer) and processed with ImageJ Software for each of the three independent experimental replicates [32 (link)].