Click-iT® EdU Imaging Kits Protocol

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

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

Multicolor imaging with the Click-iT® EdU Imaging Kits.