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Copper-Catalyzed Cell Labeling

Materials Required

  • Alkyne or azide labeled cells
  • Alkyne or alkyne detection reagent
  • Copper (II) Sulfate pentahydrate
  • Aminoguanidine hydrochloride
  • PBS buffer (pH 7.4)
  • Fixative (e.g., 3.7% formaldehyde in PBS)
  • Optional: Hoechst 33342, DAPI, coverslips/microscope slides, mounting media

Material Preparation

Alkyne/Azide detection reagent, stock solutionPrepare 2 mM stock solution in DMSO or water
50x Copper/THPTA solution (2.5 mM CuSO4, 12.5 mM THPTA)Weigh out 31 mg of Copper (II) Sulfate Pentahydrate and 270 mg of THPTA, mix, add 50 mL of water, vortex to dissolve completely
50x Aminoguanidine solution (50 mM)Weigh out 55.2 mg of Aminoguanidine hydrochloride, add 10 mL of water, vortex to dissolve completely
50x Sodium Ascorbate solution (125 mM)Weigh out 20 mg of sodium ascorbate, add 2 mL of water, vortex to dissolve completely. Sodium ascorbate solution is susceptible to oxidation. We recommend always using freshly prepared solution of sodium ascorbate
Click Cocktail (50 µM CuSO4, 250 µM THPTA, 25 µM Detection Reagent, 1 mM Aminoguanidine, 2.5 mM sodium ascorbate in PBS)For 1 ml of Click Cocktail, mix 20 µL of 50x Copper/THPTA, 20 µL of 50x aminoguanidine, 20 µL of 50x Sodium Ascorbate, 12.5 µL of detection reagent stock solution, 927.5 µL of PBS.

Note: Prepare Click Cocktail 10 minutes prior to use. Chill on ice for at least 10 minutes before adding to the cells.

Cell labeling

Growth medium, cell density, cell type variations, and other factors may influence labeling. Investigators are encouraged to determine the optimal concentration of the azide-labeling reagent as well as labeling time individually for each cell type on a small-scale first. Metabolic labeling should be carefully assessed for each cell line of interest.

1. Cell Labeling with Alkyne/Azide Detection Reagent

  1. Wash azide or alkyne metabolically labeled cells twice with ice-cold PBS
  2. Add ice-cold Click Cocktail to the cells
  3. Incubate for 1-5 min on ice or at 4°C
  4. Wash the cells twice with PBS
  5. Cells are ready for imaging

2. Cell Fixation and Permeabilization (optional)

    1. Fix the cells with fixative for 10 min at room temperature
    2. Remove the fixative and wash the cells 3 times with PBS
    3. (Optional) Stain nuclei with Hoechst or DAPI
    4. Wash the cells 3 times with PBS
    5. Proceed to imaging

If dual labeling is desired: Wash the cells twice with growth media after Step 1.5. Return the cells to growth media containing azide-labeling reagent for 20 h

Selected References:
  1. Finn, M. G, et al. (2010). Labeling live cells by copper-catalyzed alkyne–azide click chemistry. Bioconjug Chem., 21 (10), 10912-6. [PubMed]