Click-iT EdU Protocol

Cell Cycle Analysis Using EdU Staining

An Advanced Method Giving You Results Superior to BrdU

“Click Chemistry” won Nobel prize for Chemistry in 2022.

This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT™ Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor™ dye. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population.

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies

The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT™ Plus detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it can be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution, as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use Click-iT™ Plus EdU assay is compatible with cell cycle dyes. The Click-iT™ Plus EdU assay can also be multiplexed with antibodies against surface and intracellular markers, as well as conjugates labeled with standard fluorophores including R-PE, R-PE tandems, and fluorescent proteins (GFP and mCherry).

In this protocol we are going to use Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay from Thermo Fisher (Catalog number: C10632). This assay is optimized for HEK cells, users have to optimize the assay for other cells. The EdU Ab is tagged with Alexa Fluor 488 and for the counter stain we will use DAPI.

Click-iT EdU Protocol (Optimized for HEK293)

  • 10mM EdU solution (EdU powder in DMSO) is stored at -20, use 1:1000 for final 10uM directly in media
  • 10X Click-iT permeabilization and wash reagent (Component E) is to be diluted 1:10 in 1% BSA in PBS before use and can be stored diluted for 6 months
  • 10X EdU Buffer Additive (Component G) is stored at -20 and is to be diluted 1:10 with deionized water before use
  • Be gentle with cells
  • Make 10mL 1% BSA in PBS per sample: 6mL will be used by itself, 3.6mL will be used to dilute 10X permeabilization wash; Do not make reaction cocktail until fixative incubation (use within 15 minutes).
  • Count and seed desired cells in dishes the day before EdU addition. 5 x 105 cells per dish recommended for HEK293. Control plates for unstained cell, DAPI only cells, Alexa488 only cells, and any other fluorophore analyzed should be plated in separate dishes.
  • Do not wash cells before adding EdU solution to media. Incubate for 1 hour (this will vary between cell types and goal). Replace with fresh media and incubate another 3 hours (variable, you can skip if necessary).
  • Harvest cells with trypsin (or any other method), quench with media, and spin down. Remove supernatant.
  • Wash cells with 3 mL 1% BSA in PBS, spin at 500g, 5 min, 4oC, remove supernatant.
  • Dislodge pellet, add 100uL Click-iT Fixative (Component D), mix, and incubate 15 minutes in the dark at room temperature. During this incubation, the Click-iT Plus Reaction Cocktail can be prepared.
  • Add 3 mL 1% BSA in PBS directly to the suspension to wash, spin at 500g, 5 min, 4oC, remove supernatant.
  • Dislodge pellet, add 100uL 1X Click-iT permeabilization and wash reagent (diluted Component E) and mix. Cells can be stored in the dark at 4°C overnight at this step.
  • Add 500 uL of the Click-iT Plus Reaction Cocktail to suspension, mix, and incubate 30 minutes in the dark at room temperature.
  • Add 3 mL 1X Click-iT permeabilization and wash reagent directly to the suspension to wash, spin at 500g, 5 min, RT, remove supernatant.
  • Dislodge pellet, add 250uL 1X Click-iT permeabilization and wash reagent to wash. Then add 5 uL of 10ug/mL DAPI directly to suspension and incubate for 2 minutes before flow cytometry.

This protocol was generously provided by Emily Satterwhite of Mansfield lab, Department of Biochemistry and Molecular Biology, BSOM.