image of a Click-iT® EdU Imaging Kit

DNA synthesis–based cell proliferation assay

In this assay, the modified thymidine analog EdU (5-ethynyl-2'-deoxyuridine, a nucleoside analog of thymidine) is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor® dye in a fast, highly-specific, mild click reaction

This protocol can be used for:

  • Detecting DNA synthesis using a fluorescence microscope

This protocol should not be used for:

You will need the following for this protocol:

Protocol

Prepare stock solutions

  1. Allow vials to warm to room temperature
  2. Add 2 mL DMSO (Component C) or an aqueous solution to Component A to make a 10 mM EdU stock solution. Store at –20°C
  3. Make 1X Click-iT® EdU reaction buffer by adding 36 mL of deionized water to Component D. Store at 2–8° C
  4. Make Alexa Fluor® azide by adding 70 μL DMSO (Component C) to Component B, then mixing well. Store at –20°C
  5. Make 10X Click-iT® EdU buffer additive by adding 2 mL deionized water to Component F and mixing. Store at –20°C
  6. Dilute Hoechst® 33342 (Component G) 1:2,000 in PBS to obtain a 1X solution

Label cells with EdU

  1. Plate cells on coverslips and incubate overnight
  2. Dilute 10 µL of 10 mM EdU stock solution in 5 mL of prewarmed tissue culture medium to make a 20 µM EdU labeling solution (enough for 10 coverslips)
  3. Remove half of the medium from cells
  4. Replace with an equal volume of EdU labeling solution (final concentration of 10 µM)
  5. Incubate cells under appropriate growth conditions and treatments for two hours. Slow-growing cells may require a longer incubation time
  6. Proceed immediately to fixation and permeabilization

Fix and permeabilize cells

  1. Transfer each coverslip into one well of a 6-well plate
  2. Add 1 mL of 3.7% formaldehyde in PBS to each well
  3. Incubate for 15 minutes at room temperature
  4. Remove formaldehyde and wash twice with 1 mL of 3% BSA in PBS
  5. Remove wash solution and add 1 mL of 0.5% Triton® X-100 in PBS to each well
  6. Incubate for 20 minutes at room temperature

 

Protocol tips

  • For in vivo experiments, additional EdU can be purchased separately (Cat. Nos. A10044, E10187)
  • Fixation/permeabilization reagents such as methanol and saponin can be used instead of the included Triton® X-100

image of cells illustrating Click-iT® labeling technology
Multicolor imaging with the Click-iT® EdU Imaging Kits.

Detect EdU

  1. Make 1X Click-iT® EdU buffer additive by diluting the 10X solution created above 1:10 in deionized water. Use this solution within 8 hours
  2. Prepare Click-iT® reaction cocktail according to the table below. Add ingredients in the order listed in the table

    Reaction components*
    Number of coverslips
      1 2 4 5 10 25 50
    1X Click-iT® EdU reaction buffer 430 µL 860 µL 1.8 mL 2.2 mL 4.3 mL 10.7 mL 21.4 mL
    CuSO4 (Component E) 20 µL 40 µL 80 µL 100 µL 200 µL 500 µL 1 mL
    Alexa Fluor® azide 1.2 µL 2.5 µL 5 µL 6 µL 12.5 µL 31 µL 62 µL
    1X Click-iT® EdU buffer additive 50 µL 100 µL 200 µL 250 µL 500 µL 1.25 mL 2.5 mL
    Total volume 500 µL 1 mL 2 mL 2.5 mL 5 mL 12.5 mL 25 mL
    *Note: Add the ingredients in the order listed in the table.
  3. Remove permeabilization buffer from cells and wash twice with 1 mL of 3% BSA in PBS
  4. Remove the wash solution
  5. Add 0.5 mL of Click-iT® reaction cocktail to each well. Rock the plate briefly to ensure even distribution of reaction cocktail
  6. Incubate the plate for 30 minutes at room temperature, protected from light
  7. Remove the reaction cocktail and wash each well once with 1 mL of 3% BSA in PBS

Additional labels

  1. Perform antibody labeling of the samples at this time
  2. Wash each well with 1 mL of PBS. Remove the wash solution
  3. Add 1 mL of 1X Hoechst® 33342 solution per well
  4. Incubate for 30 minutes at room temperature, protected from light
  5. Remove the Hoechst® 33342 solution
  6. Wash each well twice with 1 mL of PBS
  7. Remove the wash solution

Image

  1. Cells labeled with Click- iT® EdU are compatible with all methods of slide preparation including wet mount or prepared mounting media
  2. Image cells with appropriate filters listed below

      Hoechst® 33342 Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 594 Alexa Fluor® 647
    Excitation/Emission (in nm) 350/461 495/519 555/565 590/615 650/670
    Standard filter set DAPI FITC RFP TRITC Cy®5