Alexa fluor 568

Coverslips in 6-well tissue culture plates were treated with 1× poly-D lysine (Corning) solution for 3 h at 37 °C. HEK293T cells were seeded at densities of 2.5×10⁵ and grown to ~50% confluency on glass cover slips. Cells with or without ectopic expression of UCK2 were treated with nucleosides to final concentrations of 1 mM and incubated for 5 hours. After labeling, cells were washed twice with DPBS and fixed for 10 min at room temperature with 3.7% paraformaldehyde. Cells were quenched with 50 mM glycine, DPBS for 5 min, and then washed twice more. Cells were permeabilized in 0.1% Triton-X DPBS for 15 min, and then washed ×2 with DPBS. Cells were then treated with solutions containing 500 μM CuSO₄, 2.5 mM THPTA (Sigma), 2.5 mM sodium ascorbate, and 15 μM Alexa-Fluor 568 (Click Chemistry Tools) and incubated at room temperature for 1 h in the dark. Cells were washed ×2 with 0.1% Triton-X DPSB for 2 min, and then ×5 with DPBS for 5 min each on an orbital shaker. Cells were stained with a solution of 1:3000 Hoechst 333242 for 10 min (Thermo Fisher). Coverslips were briefly washed and then mounted using VectaShield mounting medium (Vector Labs). Slides were imaged via fluorescence confocal microscopy using a 63× oil immersion objective on a Leica 700 Carl Zeiss microscope.