Fig. 5. Imaging apoptotic cells with tetrazine-Cy3 after pre-targeting with bioconjugate C2Am-11. (a) Scheme of the general procedure for imaging cells. (b) Fluorescent images of non-apoptotic (control) and apoptotic HeLa cells after labelling with C2Am-11 + Tz-Cy3. Blocking studies were performed by pre-incubating of apoptotic cells with an excess of non-fluorescent C2Am before incubation with C2Am-11. (c) MFI from apoptotic and blocked cells determined with ImageJ (average of 30 regions of interest). Statistical significance was determined by paired t test by using GraphPad Prism (****, P < 0.0001). (d) Treatment of C2Am-11 with 6-methyl-tetrazine-sulfo-Cy3 gave a new fluorescent band as detected by SDS-PAGE (lanes 2 and 4) that is consistent with the fluorogenic tetrazine bonding to the azanorbornadiene. Control lanes 1 and 3 refer to incubation of unmodified C2Am with Tz-Cy3 under the same conditions. Lanes 1 and 2, Spyro Protein Ruby staining; lanes 3 and 4 fluorescence from excitation/emission of Cy3.