Copper-Catalyzed Cell Labeling
Materials Required
- Alkyne or azide labeled cells
- Alkyne or alkyne detection reagent
- Copper (II) Sulfate pentahydrate
- THPTA
- 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 solution | Prepare 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
- Wash azide or alkyne metabolically labeled cells twice with ice-cold PBS
- Add ice-cold Click Cocktail to the cells
- Incubate for 1-5 min on ice or at 4°C
- Wash the cells twice with PBS
- Cells are ready for imaging
2. Cell Fixation and Permeabilization (optional)
- Fix the cells with fixative for 10 min at room temperature
- Remove the fixative and wash the cells 3 times with PBS
- (Optional) Stain nuclei with Hoechst or DAPI
- Wash the cells 3 times with PBS
- 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:
- Finn, M. G, et al. (2010). Labeling live cells by copper-catalyzed alkyne–azide click chemistry. Bioconjug Chem., 21 (10), 10912-6. [PubMed]
