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