image of a Click-iT® EdU HCS Assays 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 high-content imager

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. Make 1X Click-iT® EdU reaction buffer by diluting Component C 1:10 with deionized water. Store at 2–8˚C
  3. Make 10X Click-iT® EdU buffer additive by adding 2 mL deionized water to Component E and mixing. After use, store remaining solution at –20°C
  4. Dilute HCS NuclearMask™ Blue stain (Component G) 1:2,000 in PBS to obtain a 1X solution

Label cells with EdU

  1. Plate cells in 96-well plates and incubate overnight
  2. Dilute 20 µL of 10 mM EdU stock solution in 10 mL of prewarmed tissue culture medium to make a 20 µM EdU labeling solution
  3. Add 100 µL of this labeling solution to each well containing treated cells in 100 µL of complete medium (10 µM final concentration)
  4. Incubate cells under appropriate growth conditions and treatments for two hours. Slow-growing cells may require a longer incubation time
  5. Proceed immediately to fixation and permeabilization

 

Protocol tips

  • Fixation/permeabilization reagents such as methanol and saponin can be used instead of the included Triton® X-100
  • For a negative staining control, include cells from the same population, but do not treat with EdU

Fix and permeabilize cells

  1. Remove medium and add 100 μL of 3.7% formaldehyde in PBS to each well
  2. Incubate for 15 minutes at room temperature
  3. Remove fixative and wash twice with PBS
  4. Remove wash solution and 100 μL of 0.1% Triton® X-100 in PBS to each well
  5. Incubate for 15 minutes at room temperature

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 table below. Add ingredients in the order listed in the table

    Reaction components* Number of plates
      0.5 1 2 5 10
    1X Click-iT® EdU reaction buffer
    5.1 mL 10.3 mL 20.6 mL 51.5 mL 103 mL
    CuSO4 (Component D) 240 μL 480 μL 960 μL 2.4 mL 4.8 mL
    Alexa Fluor® azide (Component B) 15 μL 30 μL 60 μL 150 μL 300 μL
    1X Click-iT® EdU buffer additive
    600 μL 1.2 mL 2.4 mL 6 mL 12 mL
    Total volume 6 mL 12 mL 24 mL 60 mL 120 mL
    *Note: Add the ingredients in the order listed in the table.
  3. Remove permeabilization buffer from cells and wash twice with PBS
  4. Remove the wash solution
  5. Add 100 μL Click-iT® reaction cocktail to each well
  6. Incubate for 30 minutes at room temperature, protected from light
  7. Remove the reaction cocktail and wash each well once with 100 μL of Click-iT® reaction rinse buffer (Component F)

Additional labels

  1. Perform antibody labeling of the samples at this time
  2. Wash each well with PBS. Remove the wash solution
  3. Dilute 2 μL HCS NuclearMask™ Blue stain (Component G) in 4 mL PBS to obtain a 1X working solution
  4. Remove any wash solution from cells
  5. Add 100 μL of 1X HCS NuclearMask™ Blue stain solution to each well
  6. Incubate for 30 minutes at room temperature, protected from light
  7. Remove the HCS NuclearMask™ Blue stain solution
  8. Wash each well twice with PBS
  9. Remove the wash solution

Image

  1. Add PBS to each well. Seal the plate with sealing film, if desired
  2. Cells labeled with Click- iT® EdU are compatible with all methods of slide preparation including wet mount or prepared mounting media
  3. Image cells with appropriate filters listed below

      HCS NuclearMask™ Blue stain 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