EdU-based kits used for detecting proliferating cells were first commercialized by ThermoFisher Scientific and sold under Click-iT^® EdU assays. As with many commercial kits, they provide great results but are often quite expensive and provide fixed amounts of reagents, which limits optimized or off-protocol use of these kits. Self-assembled 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.

Materials Required

Material Preparation

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. Suspend the cells in an appropriate culture medium to obtain optimal conditions for cell growth. Disturbing the cells by temperature changes or washing prior to incubation with EdU slows the growth of the cells during incorporation.
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). We have found a final concentration of 10 μM EdU to be sufficient for labeling of most cell lines
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 1-2 hours
   Altering the amount of time the cells are exposed to EdU or subjecting the cells to pulse labeling with EdU allows the evaluation of various DNA synthesis and proliferation parameters. Effective time intervals for pulse labeling and the length of each pulse depend on the cell growth rate
6. Proceed immediately to Cell fixation and permeabilization or cell-surface antigen staining with antibodies (optional)
7. After EdU labeling, wash cells once with 3 mL of 1% BSA in PBS, pellet cells by centrifugation, and remove supernatant
8. Dislodge the pellet and resuspend cells at 1 × 10⁷ cells/mL in 1% BSA in PBS
9. Add surface antibodies, mix well and incubate for the recommended time and temperature while protected from light
10. Proceed to cell fixation step

2. Cell fixation and permeabilization

The following protocol is provided for the fixation step using saponin-based permeabilization and wash reagent. This reagent can be used with whole blood or cell suspensions containing red blood cells, as well as with cell suspensions containing more than one cell type. This permeabilization and wash reagent maintains the morphological light scatter characteristics of leukocytes while lysing red blood cells. Protocols using other fixation/permeabilization reagents, such as methanol followed by a 0.5% Triton^® X-100 permeabilization step, can also be used.

1. Wash the cells once with 3 mL of 1% BSA in PBS, pellet the cells, and remove the supernatant
2. Dislodge the pellet, add 100 μL of Fixative and mix well, and incubate for 15 minutes
3. Remove the fixative and wash the cells in each with 1 mL of 1% BSA in PBS. If red blood cells or hemoglobin are present in the sample, repeat the wash step. If required, remove all residual cell debris
4. Remove the wash solution and resuspend the cells in 100 μL of saponin-based permeabilization and wash reagent, and mix well. Incubate the cells for 15 minutes
5. Proceed directly to EdU detection step or to treating cells with antibodies to intracellular antigens followed by the EdU detection step

3. EdU detection

Note: 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 compo­nent	Number of coverslips
   	1	2	5	10	15	50
   PBS Buffer	438 µL	875 µL	2.19 mL	4.38 mL	6.57 mL	21.9 mL
   Copper Sulfate (Material preparation)	10 µL	20 µL	50 µL	100 µL	150 µL	500 mL
   AZDye Azide Solution (Material preparation)	2.5 µL	5 µL	12.5 µL	25 µL	37.5 µL	125 µL
   Reducing Agent (Material preparation)	50 µL	100 µL	250 µL	500 µL	750 µL	2.5 mL
   Total Volume	500 µL	1 mL	2.5 mL	5 mL	7.5 mL	25 mL

2. Remove the permeabilization buffer (step 2.4)
3. Add 0.5 mL of the Reaction Cocktail to each tube and mix
4. Protect from light, and incubate the plate for 30 minutes at room temperature
5. Remove the reaction cocktail. Wash the cells once with 3 mL of saponin-based permeabilization and wash reagent. Pellet the cells and remove the wash solution
6. If intracellular antibody labeling is required, dislodge the cell pellet and resuspend the cells in 100 μL of saponin-based permeabilization and wash reagent
7. If no intracellular antibody labeling is required, add 500 μL of saponin-based permeabilization and wash reagent
   At this point the samples are ready for DNA staining. If no DNA staining is desired, proceed to analyzing the cells on a flow cytometer

4. (Optional) intracellular or surface antigens staining

1. Add antibodies against intracellular antigens or against surface antigens that use RPE, PR-tandem or Quantum Dot antibody conjugates. Mix well
2. Protect from light and incubate the tubes for the time and temperature required for antibody staining
3. If EdU Detection has not yet been performed, wash the cells once with 3 mL of 1% BSA in PBS, pellet the cells, and remove the supernatant. Dislodge the cell pellet and add 100 μL of saponin-based permeabilization and wash reagent, mix well and proceed to EdU Detection (step 3.1)
4. If EdU Detection has been performed (steps 3.1–3.7), wash each tube with 3 mL of saponin-based permeabilization and wash reagent, pellet the cells, and remove the supernatant. Dislodge the cell pellet and resuspend the cells in 500 μL of saponin-based permeabilization and wash reagent, and proceed to DNA staining, or to the cell analysis on a flow cytometer

5. DNA staining

1. Add the appropriate DNA stain to each tube and mix well.
2. Protect from light and incubate as recommended by manufacturer
3. Remove the DNA stain and wash each tube with 3 mL of saponin-based permeabilization and wash reagent

Flow Cytometry Analysis

Use a low flow rate during acquisition, if a traditional flow cytometer with a hydrodynamic focusing is used to measure the total DNA content. The same collection rate and cell concentration should be used for each sample within an experiment. Detect the fluorescent signal generated by DNA content stains with linear amplification. The fluorescent signal generated by EdU labeling is best detected with logarithmic amplification.
The excitation and emission maxima of the available dyes are listed in table 2.

Table 2