Click-iT® EdU HCS Assays Protocol | Thermo Fisher Scientific

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:

Flow cytometry; for a flow cytometry protocol see Click-iT® Plus Assay Kits for Flow Cytometry

You will need the following for this protocol:

Click-iT® EdU HCS Kit (Cat. Nos. C10350, C10351, C10352, C10353, C10354, C10355, C10356, C10357)
PBS (Cat. No. 10010-023)
Fixative (e.g., 3.7% Formaldehyde in PBS, (Cat. No. R37602))
Permeabilization reagent (e.g., 0.5% Triton® X-100 in PBS (Cat. No. R37602))
3% bovine serum albumin (BSA) in PBS (3% BSA in PBS), pH 7.4
Deionized water
96-well plates

Protocol

Prepare stock solutions

Allow vials to warm to room temperature
Make 1X Click-iT® EdU reaction buffer by diluting Component C 1:10 with deionized water. Store at 2–8˚C
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
Dilute HCS NuclearMask™ Blue stain (Component G) 1:2,000 in PBS to obtain a 1X solution

Label cells with EdU

Plate cells in 96-well plates and incubate overnight
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
Add 100 µL of this labeling solution to each well containing treated cells in 100 µL of complete medium (10 µM final concentration)
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

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

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

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 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.

Remove permeabilization buffer from cells and wash twice with PBS
Remove the wash solution
Add 100 μL Click-iT® reaction cocktail to each well
Incubate for 30 minutes at room temperature, protected from light
Remove the reaction cocktail and wash each well once with 100 μL of Click-iT® reaction rinse buffer (Component F)

Additional labels

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

Image

Add PBS to each well. Seal the plate with sealing film, if desired
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

	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