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EdU Plus Cell Proliferation Assay Protocol for Fluorescent Microscopy

Many commercial EdU-based kits used for detecting proliferating cells provide great results, but are often expensive and provide fixed amounts of reagents, which limits optimized or off-protocol use of these kits. “Home-made” kits are a viable alternative to commercially available kits, in particular when all of the components are widely available from a number of suppliers. The amounts of reagents and the click reaction conditions are very similar between many commercial kits, and are in line with large number of published procedures for EdU-based detection of proliferating cells. Using the provided protocols, a researcher will be able to assemble an EdU-based cell proliferation assay that would require very little, if any, fine tuning.

EdU Plus kits with improved biocompatibility and detection limits were first commercialized by ThermoFisher Scientific and sold under Click-iT® Plus EdU assays label. The introduction of a copper-chelating moiety at the azide reporter molecule allows for a dramatic increase of the effective Cu(I) concentration at the reaction site, enhancing the weakest link in the reaction rate acceleration, greatly increasing the sensitivity biocompatibility of EdU-based assays for analyzing DNA replication in proliferating cells.

Materials Required

  • EdU Reagent
  • AZDye Picolyl Azide
  • Copper (II) Sulfate pentahydrate
  • THPTA
  • Sodium ascorbate
  • Fixative (3.7% formaldehyde in PBS)
  • Permeabilization reagent (for example, 0.5% solution of Triton®X-100 in PBS)
  • 3% BSA in PBS (pH 7.4)
  • Coverslips/microscope slides
  • Mounting media
  • Hoechst 33342 (optional)

Material Preparation

EdU Stock SolutionPrepare 20 mM solution of EdU in DMSO or water, for example to make 1 mL of 20 mM stock solution dissolve 5 mg in 1 mL of DMSO or Water
AZDye Picolyl Azide Stock SolutionPrepare 2 mM solution in DMSO or water. Example: to make 70 µL, dissolve the entire AZDye Picolyl Azide Kit Pack in 70 µL of DMSO or water
Copper Catalyst (25 mM CuSO4, 62.5 mM THPTA) solutionWeight out 312 mg of Copper (II) Sulfate Pentahydrate and 1.35 g of THPTA, add 50 mL of water, vortex to dissolve completely
Reaction Buffer50 mM Tris, 150 mM NaCl, pH 7.5. Dissolve 3.02 g of Tris, 4.4 g of NaCl in 500 mL of water, adjust pH to 7.5, sterile filter
Hoechst 3334210 mg/mL stock solution. Dissolve 1 mg of Hoechst 33342 in 100 µL of deionized water
Reducing AgentDissolve 20 mg of sodium ascorbate in 1.8 mL of deionized water. Vortex until completely dissolved. Sodium ascorbate solution is susceptible to oxidation. We recommend always using freshly prepared solution of sodium ascorbate
Wash buffer0.5 mM EDTA, 2 mM NaN3 in PBS. Add 1 mL of 0.5 M EDTA and 0.13 g of dry sodium azide to 1 L of PBS. Sterile filter for long term storage

1. Cell labeling with EdU

This protocol is based on a large number of publications of EdU-based procedures for analyzing DNA replication in proliferating cells used with different types of cells. An optimized EdU concentration is around 10 μM, but may need slight adjustment depending on the given cell type. Growth medium, cell density, cell type variations, and other factors may influence labeling. Investigators are encouraged to determine the optimal concentration of the EdU reagent as well as labeling time individually for each cell type on a small-scale first. Metabolic labeling is a critical step for successful cell proliferation and should be carefully assessed for each cell line of interest.

  1. Plate the cells on coverslips at the desired density and allow them to recover overnight before additional treatment
  2. Prepare 20 mM solution of EdU in DMSO or water
  3. Add desired amount of EdU to cells in culture medium to achieve optimal working EdU concentration (10 μM, if not optimized)
  4. During addition of EdU to cells in culture, avoid disturbing the cells in ways that may disrupt the normal cell cycling patterns
  5. Incubate the cells for the desired length of time under conditions optimal for the cell type. Different cell types may require different incubation periods for optimal labeling with EdU. As a starting point we recommend 10 μM EdU for 1 hour
  6. Proceed immediately to Cell fixation and permeabilization

2. Cell fixation and permeabilization

The following protocol is provided for the fixation step using 3.7% formaldehyde in PBS followed by a 0.5% Triton® X-100 permeabilization step. Protocols using other fixation/permeabilization reagents, such as methanol and saponin, can also be used.

  1. Transfer each coverslip into a single well. For convenient processing, use 6-well plates
  2. After metabolic labeling, remove the media and add 1 mL of 3.7% formaldehyde in PBS to each well containing the coverslips. Incubate for 15 minutes at room temperature
  3. Remove the fixative and wash the cells in each well twice with 1 mL of 3% BSA in PBS
  4. Remove the wash solution. Add 1 mL of 0.5% Triton® X-100 in PBS to each well, then incubate at room temperature for 20 minutes

3. EdU detection

Note: 500 μL of the reaction cocktail is used per coverslip. A smaller volume can be used as long as the remaining reaction components are maintained at the same ratios.

  1. Prepare the required amount of the reaction cocktail according to Table 1. Add the ingredients in the order listed in the table. Use the reaction cocktail within 15 minutes of preparation.

    Table 1

    Reaction compo­nentNumber of coverslips
    1245102550
    Reaction Buffer
    (Material preparation)
    430 µL860 µL1.7 mL2.2 mL4.3 mL10.7 mL21.4 mL
    Copper Catalyst
    (Material preparation)
    20 µL40 µL80 µL100 µL200 µL500 µL1 mL
    Picolyl Azide Solution
    (Material preparation)
    1.2 µL2.5 µL5 µL6 µL12.5 µL31 µL62 µL
    Reducing Agent
    (Material preparation)
    50 µL100 µL200 µL250 µL500 µL1.25 µL2.5 mL
    Total Volume500 µL1 mL2.0 mL2.5 mL5.0 mL12.5 mL25 mL
  2. Remove the permeabilization buffer (step 2.4). Wash the cells in each well twice with 1 mL of 3% BSA in PBS. Remove the wash solution.
  3. Add 0.5 mL of the Reaction Cocktail to each well containing a coverslip. Rock the plate briefly to ensure that the reaction cocktail is distributed evenly over the coverslip.
  4. Protect from light, and incubate the plate for 30 minutes at room temperature
  5. Remove the reaction cocktail. Wash each well once with 1 mL of 3% BSA in PBS. Remove the wash solution.
  6. Wash each well once with 1 mL of Wash Buffer. Remove the wash solution.
  7. Wash each well once with 1 mL of PBS. Remove the wash solution.
    At this point the samples are ready for DNA staining. If no DNA staining is desired, proceed to Imaging
    If antibody labeling of the samples is desired, proceed to labeling according to manufacturer’s recommendations. Keep the samples protected from light during incubation.

4. DNA staining

  1. Wash each well with 1 mL of PBS. Remove the wash solution.
  2. repare 1x Hoechst 33342 solution by diluting stock solution of Hoechst 33342 1:2000. The final concentration of 1x Hoechst 33342 solution is 5 µg/mL.
    Final concentrations of 1x Hoechst 33342 may range from 2 μg/mL to 10 μg/mL.
  3. Add 1 mL of 1x Hoechst 33342 solution per well. Protect from light. Incubate for 30 minutes at room temperature.
  4. Remove the Hoechst 33342 solution.
  5. Wash each well twice with 1 mL of PBS.
  6. Remove the wash solution.

Imaging

Labeled cells are compatible with all methods of slide preparation