Click-iT EdU Imaging Cell Proliferation Protocol

The Click-iT EdU Imaging Kits Protocol presents an alternative approach to DNA synthesis-based cell proliferation assays. Unlike traditional BrdU protocols, which rely on antibody-based detection methods, the Click-iT EdU protocol utilizes a modified thymidine analog, EdU (5-ethynyl-2'-deoxyuridine) labeling with click chemistry. This nucleoside analog is efficiently incorporated into newly synthesized DNA and subsequently labeled with a bright, photostable Alexa Fluor dye through a fast, highly-specific, mild click reaction. The Click-iT EdU kit simplifies the detection process, offering enhanced specificity and reduced background staining compared to conventional EdU assays. These improvements make it an invaluable tool for researchers evaluating cell proliferation, facilitating accurate and reliable analysis in various applications.

See Click-iT EdU cell proliferation assays

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Materials
Protocol
Ordering information
Resources

Materials

This protocol can be used for:

Detecting DNA synthesis using a fluorescence microscope

This protocol should not be used for:

Flow cytometry; for a flow cytometry protocol, see Click-iT EdU Protocol for Flow Cytometry

You will need the following for this protocol:

Click-iT EdU Imaging Kit (Cat. Nos. C10338, C10340, C10337, C10339)
PBS
Fixative (e.g., 3.7% Formaldehyde in PBS)
Permeabilization reagent (e.g., 0.5% Triton X-100 in PBS)
3% bovine serum albumin (BSA) in PBS (3% BSA in PBS), pH 7.4
Deionized water
18 x 18-mm coverslips
Optional: 6-well microplate

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Protocol steps

Prepare stock solutions

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

Label cells with EdU

Plate cells on coverslips and incubate overnight.
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).
Remove half of the medium from cells.
Replace with an equal volume of EdU labeling solution (final concentration of 10 µM).
Incubate cells under appropriate growth conditions and treatments for two hours. Slow-growing cells may require a longer incubation time.
Proceed immediately to fixation and permeabilization.

Fix and permeabilize cells

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

Detect EdU

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.

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.

Remove permeabilization buffer from cells and wash twice with 1 mL of 3% BSA in PBS.
Remove the wash solution.
Add 0.5 mL of Click-iT reaction cocktail to each well. Rock the plate briefly to ensure even distribution of reaction cocktail.
Incubate the plate for 30 minutes at room temperature, protected from light.
Remove the reaction cocktail and wash each well once with 1 mL of 3% BSA in PBS.

Additional labels

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

Imaging

Cells labeled with Click- iT EdU are compatible with all methods of slide preparation including wet mount or prepared mounting media.
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 Cy5

	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	Cy5

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Ordering information

Click-iT EdU materials

Resources

Cell Viability, Proliferation, and Cell Cycle Information
Find educational resources for monitoring cell function

Cell Proliferation Assay Protocols
Discover protocols for various applications to study cell proliferation.

Cell Cycle & Proliferation Pathways
Investigate signaling pathways and signal transduction.

Fluorescence SpectraViewer
Use SpectraViewer to easily compare excitation and emission spectra of fluorophores and reagents.