Fig. 4.

Picolyl-containing azide reporters generally increase the sensitivity of alkyne lipid imaging. Quantification of signal intensities of alkyne lipids in HuH7 cells labeled with different azide detection reagents: HuH7 cells were incubated with medium supplemented with 2.5 μM alkyne-oleate, alkyne-cholesterol, or propargylcholine, or in medium without lipid supplementation for 16 h. After fixation, cells were click-labeled using 10 μM azide detection reagent and 200 μM CuTFB. Epifluorescence images were taken with 50 ms exposure, a high dynamic range camera (Orca-Flash4.0) and an Apochromate 63× (1.40 NA) DIC Oil objective (excluding the edges of the field of view to assure even illumination) and stitched to one large image of 1,000 × 1,000 μm for every sample. In these images, the mean signal of 80 to 160 cells was determined as described in Fig. 3E. Data points correspond to the mean signal of individual cells. In addition, the mean value and the standard deviation for all cells of a sample are given in the graph. Asterisks designate levels of statistical significance (one-way ANOVA test) for a difference in the mean of a sample and its corresponding control (no lipid). Color-coding of the data corresponds to that in Figs. 1, 3, and 5.