Phospholipid and neutral lipid droplet staining

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Neutral lipid droplets and phospholipidosis were analyzed using Invitrogen´s HCS LipidTOXTM Phospholipidosis and Steatosis Detection Kit for image-based high-content screening assays. Staining was prepared following the protocol of the manufacturer. Four day M-CSF differentiated macrophages were incubated with LipidTOXTM Red to detect phospholipidosis in the presence of M-CSF, E-LDL or Ox-LDL for 24h. Afterwards cells were fixed with 4% paraformaldehyde for 20 min at room temperature and after washing with PBS stained with LipidTOXTM Green for 20 min at room temperature to detect neutral lipid droplets. Subsequently nuclei were stained using Draq5 (5μM) (Axxora, Deutschland GmbH). Two positive control compounds, propranolol which induces accumulation of phospholipids and cyclosporine A which induces formation of prominent neutral lipid droplets are included in the kit and were applied to the M-CSF differentiated, E-LDL and Ox-LDL loaded macrophages.


 

Figure 1: Neutral lipid and phospholipid staining in macrophages
M-CSF differentiated, E-LDL and Ox-LDL loaded macrophages labeled with LipidTOXTM Green neutral lipid droplet stain (green fluoresence) and LipidTOXTM Red phospholipidosis detection reagent (red fluorescence) and the corresponding overlay with the nuclei stain Draq5 (blue fluorescence). E-LDL loaded macrophages show intensive neutral lipid droplet staining with LipidTOXTM Green (neutral lipid droplet stain; green staining) compared to Ox-LDL loaded and M-CSF differentiated macrophages. In contrast Ox-LDL loaded macrophages show pronounced phospholipid staining with LipidTOXTM Red (phospholipidosis detection reagent; red staining) compared to E-LDL loaded and M-CSF differentiated macrophages. Incubation with the control substances propranolol for phospholipidosis induction and cyclosporine A for the induction of neutral lipids show the expected characteristics (images not shown).

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