image of a Click-iT EdU Imaging Kit

DNA synthesis–based cell proliferation assay

In this assay the modified thymidine analogue 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 transferring the solution (4 mL) in the Component D bottle to 36 mL of deionized water. Store any remaining solution at 2–8˚C
  4. Make 10X Click-iT EdU buffer additive by adding 2 mL deionized water to Component F and mixing until fully dissolved. Store at –20°C
  5. 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 for two hours. NOTE: the choice of incubation time depends on the cell growth rate; thus optimization may be required
  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 labeled with Click-iT Plus EdU Alexa Fluor 647 Imaging Kit
ERK2 A375 GFP expressing cells labeled with Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (pink), Proliferating cells have pink nuclei. Non-proliferating cells have blue nuclei.

Detect EdU

  1. Make 1X Click-iT EdU buffer additive by diluting the 10X solution created above 1:10 in deionized water. Prepare this solution fresh and use the solution on the same day.
  2. Prepare Click-iT Plus reaction cocktail according to the table below. IMPORTANT: add the ingredients in the order listed in the table. 


    3. Reaction components*
    Number of coverslips
     1245102550
    1X Click-iT reaction buffer440 µL880 µL1.84 mL2.25 mL4.4 mL10.9 mL21.9 mL
    Copper protectant10 µL20 µL40 µL50 µL100 µL250 µL500 µL
    Alexa Fluor picolyl azide (Component B) 1.2 µL2.5 µL5 µL6 µL12.5 µL31 µL62 µL
    1X Click-iT EdU buffer additive50 µL100 µL200 µL250 µL500 µL1.25 mL2.5 mL
    Total volume500 µL1 mL2 mL2.5 mL5 mL12.5 mL25 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 Plus 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

Stain DNA

  1. Wash each well with 1 mL of PBS. Remove the wash solution
  2. Add 1 mL of 1X Hoechst 33342 solution per well
  3. Incubate for 30 minutes at room temperature, protected from light
  4. Remove the Hoechst 33342 solution
  5. Wash each well twice with 1 mL of PBS
  6. Remove the wash solution
  7. OPTIONAL: Perform antibody labeling of the samples at this time, following the recommendations from the manufacturer of the primary and secondary antibody. Note: protected samples from light during these incubations.

Image

  1. Cells labeled with Click-iT Plus 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 33342Alexa Fluor 488Alexa Fluor 555Alexa Fluor 594Alexa Fluor 647
Excitation/Emission (in nm)350/461495/519555/565590/615650/670
Standard filter setDAPIFITCRFPTRITCCy5