logo
AAT Bioquest

Sensitive Identification of Newly Synthesized DNA In Situ using click chemistry-based Bucculite™ XdU Cell Proliferation Assays

Abstract


The measurement of cell proliferation in situ is fundamental to the assessment of cell health, genotoxicity, and drug efficacy evaluation. Precisely labeling and quantifying cells in the synthesis phase (S phase) of cell cycle progression is not only adequate for characterizing basic biology but can provide insight into cell cycle kinetics, mechanisms of cell growth, and cytotoxicity. In drug discovery, cell proliferation assays can facilitate the determination of novel therapeutics, define cellular responses to drug treatments, and evaluate the antiproliferative potency and toxicity of drug candidates. Traditionally, cell proliferation measurements rely on incorporating radioactive nucleosides, such as 3H-thymidine, into DNA or incorporating the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) during synthesis, with subsequent detection by an anti-BrdU antibody conjugate. While BrdU proliferation assays provided a safer alternative to radioactive 3H-thymidine, the harsh treatments needed to denature the DNA and facilitate the detection of the incorporated BrdU molecules by antibodies can adversely affect cell morphology and antigen recognition sites resulting in poor image quality. The Bucculite™ XdU Proliferation assays represent a significant breakthrough in the evolution of cell proliferation measurements. Unlike traditional cell proliferation assays, the Bucculite™ XdU Cell Proliferation assays are not dependent upon radioactivity or antibodies for detection. Instead, a proprietary mixture of alkyne-containing thymidine analogs, XdU, is incorporated into DNA during active DNA synthesis. The incorporated XdU is then detected by a copper-catalyzed azide-alkyne cycloaddition or click reaction using a fluorescent dye containing a picolyl azide moiety.

 

Introduction


Quantitative analysis of newly synthesized DNA in situ in the presence of a label is considered one of the most reliable and accurate assay types for measuring cell proliferation. Initially, proliferation measurements relied on the incubation of 3H-thymidine for several hours to overnight. Proliferating cells would incorporate the radioactive 3H-thymidine into their newly synthesized DNA. After subsequent washing and adherence to filters, a scintillation beta-counter is used to measure the radioactivity in DNA recovered from the cells to determine the degree of cell division. Besides a lengthy and tedious protocol, the health hazards of working with radioactive materials are well recognized, as are the accompanying economic considerations required for appropriate handling and disposal of radioactive waste.

Years later, a nonradioactive approach was introduced based on the detection of incorporated 5-bromo-2'-deoxyuridine (BrdU) by anti-BrdU antibody conjugates. Although BrdU proliferation assays eliminated the risks and complications associated with handling radioactive materials, it too is burdened by a tedious and time-consuming protocol. Assay workflow requires harsh treatments either by acid, heat, or enzymes to denature the DNA and facilitate the detection of the incorporated BrdU molecules by anti-BrdU antibody conjugates. Without DNA denaturation, BrdU is accessible to antibodies for detection. Consequently, these harsh treatments damage antigen recognition sites and adversely affect sample integrity and cell morphology. Furthermore, the denaturants used to enable antibody accessibility of the incorporated BrdU also limits its compatibility to be multiplexed with other organelle stains, fluorescent proteins, such as GFP or RFP, or phycobiliproteins, such as phycoerythrin (PE) or allophycocyanin (APC).

 

Click- Chemistry Driven Bucculite™ XdU Cell Proliferation Assays


The click chemistry-based Bucculite™ XdU Cell Proliferation Assays represents the newest addition of AAT Bioquest's growing family of proliferation reagents. Unlike traditional cell proliferation assays, Bucculite™ XdU Cell Proliferation Assays do not rely on radioactivity or antibodies to quantify nascent DNA. Rather, Bucculite™ XdU Cell Proliferation Assays use a proprietary mixture of alkyne-containing thymidine analogs, XdU, which are incorporated into newly synthesized DNA during the S phase. The incorporated XdU is subsequently detected via a copper-catalyzed azide-alkyne cycloaddition (i.e., click reaction) using various fluorophore containing a picolyl azide moiety, such as iFluor® 488 azide.

Bucculite™ XdU Cell Proliferation Assay Principle

Bucculite™ XdU Cell Proliferation Assay Principle. Proliferating cells in the presence of XdU incorporate the compound at thymidine bases during the S phase. Fluorophore-labeled azide reacts with the incorporated XdU to allow detection by imaging or flow cytometry (figure made in BioRender).


Since the click reaction utilizes biorthogonal moieties to detect proliferating cells, background interference is minimal. More importantly, because of the mild reaction conditions, Bucculite™ XdU Cell Proliferation Assays preserve cell morphology, antigen-binding sites, and sample integrity, affording the user an opportunity for multiplexing analysis. After thymidine incorporation, the click reaction and subsequent wash steps can be completed in less than 60 minutes, and nascent DNA can be quantified using standard imaging systems (Figure 2) or a flow-cytometer (Figure 3).

Microscopic Imaging of Bucculite™ XdU Cell Proliferation Assay
Microscopic imaging
Flow Cytometric Analysis of Bucculite™ XdU Cell Proliferation Assay
Flow cytometric analysis

Left: Cell proliferation detected using the Bucculite™ XdU Cell Proliferation Assay. HeLa cells were processed using the reagents and fixation/detection protocol provided in the Bucculite™ XdU Cell Proliferation iFluor® 444 Imaging kit (Cat No. 22326) and the Bucculite™ XdU Cell Proliferation iFluor® 647 Imaging kit (Cat No. 22328). Both samples were counter-stained with Hoechst® 33342 nucleic acid stain (blue, Cat No. 17530) and imaged using a fluorescence microscope. Right: Cell proliferation detected using the Bucculite™ XdU Cell Proliferation Assay. Jurkat cells were processed using the reagents and fixation/detection protocol provided in the Bucculite™ XdU Cell Proliferation iFluor® 444 Imaging kit (Cat No. 22326). Data were collected and analyzed using an ACEA Novocyte flow cytometer with 488 nm excitation and a 530/30 nm bandpass emission filter for detection of the iFluor® 488–labeled XdU.

 

Table 1. Click-chemistry based cell proliferation kits suitable for imaging or flow cytometry.

Name
Size
Cat No.
Bucculite™ Flow Cytometric XdU Cell Proliferation Assay Kit *Violet Laser-Comptatible*100 Tests22321
Bucculite™ Flow Cytometric XdU Cell Proliferation Assay Kit *Blue Laser-Comptatible*100 Tests22323
Bucculite™ Flow Cytometric XdU Cell Proliferation Assay Kit *Red Laser-Comptatible*100 Tests22325
Bucculite™ XdU Cell Proliferation Fluorescence Imaging Kit *Green Fluorescence*200 Tests22326
Bucculite™ XdU Cell Proliferation Fluorescence Imaging Kit *Red Fluorescence*200 Tests22327
Bucculite™ XdU Cell Proliferation Fluorescence Imaging Kit *Deep Red Fluorescence*200 Tests22328

 

References


  1. Buck, S. B., Bradford, J., Gee, K. R., Agnew, B. J., Clarke, S. T., & Salic, A. (2008). Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies. BioTechniques, 44(7), 927–929. https://doi.org/10.2144/000112812
  2. Clarke, S. T., Calderon, V., & Bradford, J. A. (2017). Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry. Current protocols in cytometry, 82, 7.49.1–7.49.30. https://doi.org/10.1002/cpcy.24
  3. Diermeier-Daucher, S., Clarke, S. T., Hill, D., Vollmann-Zwerenz, A., Bradford, J. A., & Brockhoff, G. (2009). Cell type specific applicability of 5-ethynyl-2'-deoxyuridine (EdU) for dynamic proliferation assessment in flow cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 75(6), 535–546. https://doi.org/10.1002/cyto.a.20712
  4. Li, X., & Darzynkiewicz, Z. (1995). Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell proliferation, 28(11), 571–579. https://doi.org/10.1111/j.1365-2184.1995.tb00045.x
  5. Salic, A., & Mitchison, T. J. (2008). A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proceedings of the National Academy of Sciences of the United States of America, 105(7), 2415–2420. https://doi.org/10.1073/pnas.0712168105