Click-iT Plus Imaging Cell Proliferation Protocol

The Click-iT Plus Plus EdU Imaging kit is a powerful tool for studying cell proliferation through DNA synthesis. Utilizing the modified thymidine analogue EdU (5-ethynyl-2′-deoxyuridine), this assay allows for the efficient incorporation of EdU into newly synthesized DNA. The incorporated EdU is then fluorescently labeled with a bright, photostable Alexa Fluor dye using a fast, highly specific, and mild click reaction. This reaction is rapid and occurs under conditions that preserve cell integrity, making it ideal for imaging cell proliferation. The use of a picolyl azide combined with a copper protectant is the basis of the upgraded Click-iT Plus EdU technology, which achieves the same sensitive, reliable detection of cell proliferation as the original Click-iT EdU assay while also preserving the fluorescence of GFP, RFP, and R-PE.

See all Click-iT EdU cell proliferation assays

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Materials
Protocol
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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 Plus EdU Imaging Kit (Cat. Nos. C10637, C10638, C10639, C10640)
PBS, pH 7.4
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 transferring the solution (4 mL) in the Component D bottle to 36 mL of deionized water. Store any remaining solution at 2–8˚C.
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.
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 for two hours.
NOTE: the choice of incubation time depends on the cell growth rate; thus optimization may be required.
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

Remove permeabilization buffer from cells and wash twice with 1 mL of 3% BSA in PBS.
Remove the wash solution.

Reaction components*	Number of coverslips
	1	2	4	10	25	50
1X Click-iT reaction buffer	440 µL	880 µL	1.84 mL	4.4 mL	10.9 mL	21.9 mL
Copper protectant	10 µL	20 µL	40 µL	100 µL	250 µL	500 µL
Alexa Fluor picolyl azide (Component B)	1.2 µL	2.5 µL	5 µL	12.5 µL	31 µL	62 µL
1X Click-iT EdU buffer additive	50 µL	100 µL	200 µL	500 µL	1.25 mL	2.5 mL
Total volume	500 µL	1 mL	2 mL	5 mL	12.5 mL	25 mL
*Note: Add the ingredients in the order listed in the table.

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.

Stain DNA

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.
Optional: Perform antibody labeling of the samples at this time, following the recommendations from the manufacturer of the primary and secondary antibody.
NOTE: Protect samples from light during these incubations.

Imaging

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