Assaying cell proliferation and DNA replication with Click Chemistry

Cell proliferation assays are important in cytotoxicity studies, cancer research, and many other areas of cell biology. A number of methods have been developed including those based on radioactivity. But most of them producing visual or fluorescent readout are also suitable for high-throughput screening.

Lumiprobe is now offering a cost-efficient set of reagents for the analysis of cell proliferation.

cell proliferation assay

Detection of replicated DNA is arguably the most direct way to detect proliferation. This has been accomplished using bromo-deoxyuridine (BrdU) nucleoside which is incorporated into DNA during replication. Then, these nucleoside modifications in cellular DNA can be detected by anti-BrdU antibodies. Although being specific, this assay is tedious and difficult to reproduce because a treatment of cells with harsh reagents is required to expose DNA to antibodies which otherwise do not penetrate cellular structures.

A milder alternative is to use ethynyl deoxyuridine (EdU) nucleoside which can be supplied to cells via feeding or injection. This nucleoside incorporates into DNA and can be then conjugated with various fluorescent dye azides under copper(I) catalysis, which provides fluorescent staining of replicated DNA. The assay is easy to perform, it is fast and reproducible. It does not require harsh treatment of the cells (only Triton permeabilization is needed), and therefore better preserves cellular structures. After the treatment with dye azide and the last washing step (step 8), the cells can be labelled using reagents containing fluorophores with different emission wavelengths such as Hoechst, DAPI, or fluorescently-labelled antibodies.

Required reagents:

  1. EdU, ethynyldeoxyuridine nucleoside
  2. Cu-TBTA complex, Click Chemistry catalyst
  3. Ascorbic acid - reductant for copper
  4. Fluorescent dye azides: Sulfo-Cyanine3 azide, Sulfo-Cyanine5 azide, BDP FL azide
  5. PBS, Triton X-100 or Tween-20

The exact protocol depends on particular cell or tissue type, but general workflow is described below:

  1. Feed cells/tissue with 10-20 uM EdU for desired time period
  2. Fix cells using 3.7% formaldehyde in PBS for 15 minutes
  3. Wash cells with PBS.
  4. Permeabilize with PBS containing 0.2% Triton X-100 or 0.5% Tween-20 for 30 min.
  5. Wash cells again with PBS.
  6. Prepare a mixture containing 2 mM Cu-TBTA complex, 5 uM dye azide, and 10 mM ascorbic acid in 100 mM Tris buffer pH 7.4 (for sulfonated azides) or 100 mM Tris buffer containing 50% DMSO (for non-sulfonated azides). This mixture should be freshly prepared, because Copper(I) solutions are unstable.
  7. Treat cells with development mixture for 30 min.
  8. Wash cells with PBS.
  9. Optional: If the cells have to be labelled with different fluorophore, proceed with staining using your standard protocol

Sulfonated, water soluble azides like Sulfo-Cyanine3 azide and Sulfo-Cyanine5 azide give the best results. When using non-sulfonated azides like Cyanine5 azide, Cyanine3 azide or BDP FL azide, ensure 50% DMSO content in development mixture. The cells can be later destained with ethanol if necessary.

References

1. Ranall, M.; Gabrielli, B.; Gonda, T. Adaptation and validation of DNA synthesis detection by fluorescent dye derivatization for high-throughput screening. BioTechniques, 2010, 48(5), 379-386. doi: 10.2144/000113410

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