Fig. 2
From: Quantitative determination of fluorescence labeling implemented in cell cultures
Application of the method to TrkA receptors labeled through Sfp. A Scheme for Sfp-based labeling applied on S6-tagged TrkA receptors expressed on living cell membranes. B Determination of labeling efficiency, performed as described in Fig. 1A, for Abberior STAR 635p (Abb.635, red channel) in conditions cB, while using two different conditions (cA on the left and \(\widetilde{{\mathrm{c}}_{\mathrm{A}}}\) on the right) for reactions with the other probe (Atto 565, orange channel). Each image column shows a single representative cell: DIC image (top) and TIRF images of the channel of the dye used in the first (middle) and second (bottom) labeling reaction. Conditions for Atto 565 were as follows: 100 nM CoA-dye, 2 µM Sfp, and 20 min reaction time (left) and 100 nM CoA-dye, 10 µM Sfp, and 10 min (right); conditions for Abb.635 were in both cases 40 nM CoA-dye, 10 µM Sfp, 20 min. r and r’ are the ratios between the number of molecules labeled in the two channels. Reported efficiency results are mean ± SEM estimate (see the “Methods” section), obtained from 17, 20, 25, and 16 cells analyzed for experiments represented in each image column. The results obtained for Abb.635 efficiency are not significantly different, and the efficiency estimated averaging the two results is 32.7 ± 1.7%. C Examples of experimental images corresponding to the workflow steps depicted in Fig. 1B, for some of the analyzed conditions reported in D. Each image column shows a single cell: DIC image (top); TIRF channel of the tested probe Atto 565 (middle, orange), TIRF image of the control probe Abb.635 (bottom, red). Eff.: efficiency estimated (for Atto 565 in varying conditions) or previously measured (for Abb.635 in fixed conditions). D Fluorescent labeling efficiency obtained for Atto 565 at various concentrations of CoA-dye ([Atto 565]) and Sfp ([Sfp]), after 20 min of reaction and in presence of 10 mM of MgCl2 (dots: mean, error bars: SEM estimates; 15–40 cells from 2 independent replicates analyzed for each condition). Scale bar: 5 μm. See also Additional file 1: Tables S1 and S2