Labeling live cells by copper-catalyzed alkyne--azide click chemistry

Abstract

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, optimized for biological molecules in aqueous buffers, has been shown to rapidly label mammalian cells in culture with no loss in cell viability. Metabolic uptake and display of the azide derivative of N-acetylmannosamine developed by Bertozzi, followed by CuAAC ligation using sodium ascorbate and the ligand tris(hydroxypropyltriazolyl)methylamine (THPTA), gave rise to abundant covalent attachment of dye-alkyne reactants. THPTA serves both to accelerate the CuAAC reaction and to protect the cells from damage by oxidative agents produced by the Cu-catalyzed reduction of oxygen by ascorbate, which is required to maintain the metal in the active +1 oxidation state. This procedure extends the application of this fastest of azide-based bioorthogonal reactions to the exterior of living cells.

Figure 1

(Top) Cell labeling steps. (Bottom) alkynyl probe reagents and catalyst additives.

Figure 2

Viability of mammalian cells after Cu-ascorbate treatment ([CuSO₄] and [THPTA] as indicated, [Na ascorbate] = 2.5 mM, DPBS, 4 °C, 5 minutes), followed by washing in buffer and incubation in growth media for 24 h. Cell viability was determined by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI).(43) (B,C,E) Cells were grown for two days in the presence or absence of Ac₄ManNAz, as indicated, before Cu treatment. Error bars indicate standard deviation from the averaging of results from at least three independent experiments.

Figure 3

Representative labeling of cells (grown in the presence of 50 μM Ac₄ManNAz for 48 h) with the indicated dye-alkynes (25 μM), in the presence of Cu catalyst (5 equiv. THPTA, 2.5 mM Na ascorbate, 1 mM aminoguanidine), in PBS pH 7.4 at 4 °C. The CuAAC reaction was conducted for 5 minutes unless otherwise noted, followed by washing in buffer and fixation for confocal microscopy imaging after incubation in fresh media for 15 minutes (except for panel D, for which the cells were incubated for 24 h before fixation and imaging). Nuclei were stained with DAPI (blue). Panels A, E, and I show the result of CuAAC labeling treatment with 1 on cells lacking the azido sugar, showing that non-specific adsorption or labeling with dye does not occur. The procedure for labeling of cells shown in panels J-L is described in the text. All images are 155 by 155 microns.

Figure 4

Flow cytometry analysis of Jurkat cells, pre-incubated for 48 h in growth medium containing 10 μM Ac₄ManNAz, and then labeled with 3 (50 μM) under standard conditions (CuSO₄ and THPTA at the indicated concentrations, 2.5 mM Na ascorbate, 1 mM aminoguanidine, in DPBS, 4 °C, 5 min). (A) All samples were analyzed in triplicate; 10,000 events each were recorded in each case, and representative histograms are shown. (B) Mean fluorescence intensities with error bars representing standard deviations (statistical analysis performed using FlowJo software 8.7.1).