Skip to main content
. Author manuscript; available in PMC: 2016 Mar 2.
Published in final edited form as: Chembiochem. 2015 Feb 6;16(4):611–617. doi: 10.1002/cbic.201402715

Figure 4. Double labeling and two-color imaging of Chol and phospholipids in cells.

Figure 4

IH-3T3 cells were incubated overnight with propargylcholine (PCho, 100 μM), to label choline phospholipids, after which they were incubated overnight in the absence or presence of eChol (40 μM from DMSO stock). EChol in cellular membranes reacts with fluorescein-azide but not with Alexa568-azide (Supplementary figure 2); this differential reactivity was used to achieve two-color imaging of eChol and PCho-labeled phospholipids. The labeled cells were first stained with Alexa568-azide (to detect PCho), after which the unreacted PCho phospholipids were consumed by reaction with excess non-fluorescent azide. Finally, the cells were stained with fluorescein-azide (to detect eChol). Top row: PCho-labeled phospholipids; second row: eChol; third row: overlay of PCho (red) and eChol (green) images; bottom row: Hoechst staining of cell nuclei.