Finally, a Proliferation Assay You Can Multiplex with GFP and R-PE
Click-iT® Plus EdU Proliferation Kits for imaging and flow cytometry platforms
Cell proliferation assays provide a critical piece of the puzzle when evaluating cell health, genotoxicity, and the efficacy of anti-cancer drugs. When compared with traditional antibody-based BrdU methods, the Click-iT® Plus EdU cell proliferation assays not only offer better performance and an easier workflow but are now compatible with an even broader range of commonly used fluorophores, including GFP and other fluorescent proteins as well as phycobiliproteins.
Choose Click-iT® Plus EdU assays over conventional BrdU assays
The most accurate cell proliferation assays directly quantitate newly synthesized DNA. Back in the 1950s, the original cell proliferation measurements were based on the incorporation of radioactive nucleosides (e.g., 3H-thymidine) into DNA. Thirty years later, this method was supplanted by the nonradioactive, antibody-based detection of the thymidine analog 5-bromo-2′-deoxyuridine (BrdU). In the standard BrdU proliferation assay, cell samples are incubated with BrdU and then treated with acid, heat, or enzymes to denature the DNA and facilitate detection of the incorporated BrdU molecules by anti-BrdU antibodies. This BrdU assay, however, is both difficult to perform consistently and time consuming, typically requiring 6–24 hr to complete. More importantly, the harsh treatments required to expose BrdU to antibody detection can adversely affect cell morphology and antigen recognition sites, as well as image quality. These denaturants also limit the ability of the BrdU proliferation signal to be multiplexed with fluorescent proteins (e.g., GFP, RFP, or mCherry) and phycobiliproteins (e.g., R-PE or R-PE tandems) that are regularly used in imaging and flow cytometry applications.
The Click‑iT® Plus EdU proliferation assay is a non-antibody alternative to the BrdU assay. In this assay, an alkyne-containing thymidine analog, EdU (5-ethynyl-2′-deoxyuridine), is incorporated into DNA during active DNA synthesis . The incorporated EdU is then detected by a click reaction (a copper-catalyzed azide–alkyne cycloaddition) using a fluorescent Alexa Fluor® dye containing a picolyl azide moiety. The click reaction and subsequent wash steps are typically completed in 60 min. Moreover, the small size of the picolyl azide dye enables efficient detection of the incorporated EdU using mild conditions that preserve GFP and R-PE fluorescence. Standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click‑iT® Plus detection reagent to gain access to the DNA; no harsh denaturants are required.
What are the advantages of Click-iT® Plus?
The Click-iT® Plus technology advances the already popular click labeling strategy by utilizing a fluorescent picolyl azide—which reacts with an alkyne such as EdU as a copper complex—along with a copper protectant to produce the same sensitive, reliable detection of cell proliferation achieved with the original Click-iT® EdU assay. The picolyl azide dyes react efficiently with complexed copper, and thus minimize the undesired copper side reactions with proteins (e.g., GFP, R-PE), nucleic acids (e.g., oligos), and even small molecules. Because of this copper protectant technology, the Click-iT® Plus assays can be used to accurately determine cell proliferation while preserving cell morphology, DNA integrity, antigen-binding sites, and GFP and R-PE fluorescence.
To demonstrate its superior performance, the Click-iT® Plus EdU assay was directly compared with the traditional BrdU assay. The proliferation signal from A375 melanoma cells expressing an Erk2-GFP fusion was detected using either BrdU or Click-iT® Plus EdU (Figure 1). The harsh treatment required for the antibody-based BrdU assay resulted in the loss of the GFP signal. Furthermore, the BrdU proliferation signal required a 10-fold longer exposure time (Figure 1B) to generate results comparable to those from the Click-iT® Plus EdU assay (Figure 1C).
|Figure 1. Cell proliferation detected using the BrdU assay or the Click-iT® Plus EdU assay. (A, B) After incubation with BrdU, Erk2-GFP–expressing A375 melanoma cells were treated with HCl, resulting in a loss of GFP signal, and incubated with Alexa Fluor® 594 anti-BrdU antibody, producing moderately bright detection of proliferation (red), shown here with (A) an 8 msec exposure and (B) an 80 msec exposure. (C) In contrast, Erk2-GFP A375 cells processed using the Click-iT® Plus EdU Alexa Fluor® 594 Proliferation Assay Kit retained their GFP signal (green), and the EdU-based detection of proliferation was very bright (red, 8 msec exposure). Both cell samples were treated with Hoechst® 33342 nucleic acid stain (blue); high-content analysis was performed using the Thermo Scientific® Cellomics® ArrrayScan® VTI HCS Reader.|
Multiplex your Click-iT® Plus EdU assays with GFP and RFP
The ability to multiplex the Click-iT® Plus EdU assays with other fluorescent probes opens the door to a more complete analysis of cell health. For example, we transduced Erk2-GFP–expressing A375 melanoma cells with CellLight® Talin-RFP. CellLight® Talin-RFP combines the BacMam 2.0 expression system with a talin-RFP fusion construct encoding a fluorescent protein that binds to the cytoskeleton. After incubation overnight with CellLight® Talin-RFP, the cells were pulsed with EdU for 2 hr and the proliferation signal was detected using the Click-iT® Plus EdU Alexa Fluor® 647 Imaging Kit (Figure 2). Using the appropriate optical filters, we simultaneously imaged the green-fluorescent Erk2-GFP fusion, expressed in the cell nuclei, and the red-fluorescent talin-RFP fusion, confirming the preservation of cytoskeleton structure. Similarly, we were able to detect in vivo DNA synthesis together with GFP expression in tissue samples from a transgenic mouse model (Figure 3).
Figure 2. GFP and RFP compatibility with the Click-iT® Plus EdU assay. Erk2-GFP–expressing A375 melanoma cells were transduced with CellLight® Talin-RFP (orange) overnight, and then pulsed with 10 µM EdU for 2 hr and labeled using the Click-iT® Plus EdU Alexa Fluor® 647 Imaging Kit (pink) and Hoechst® 33342 nucleic acid stain (blue). Proliferating cells have pink nuclei; nonproliferating cells have blue nuclei.
Figure 3. Detection of cell proliferation and GFP fluorescence in mouse tissue. A transgenic mouse was injected intraperitoneally with 50 µg EdU per gram body weight 4 hr before sacrifice. The Click-iT® Plus EdU Alexa Fluor® 555 Imaging Kit was used to detect newly synthesized DNA in mouse duodenum tissue. Constitutively expressed ϐ‑actin–GFP fusion (green) is seen in the smooth muscle band below the bright EdU-labeled proliferating cells (red) of the intestinal villi; the tissue was counterstained with DAPI nucleic acid stain (blue). Image provided by Jessica-Sordet Dessimoz, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Multiplex EdU with R-PE tandems and GFP using flow cytometry
For flow cytometry users, the new Click-iT® Plus EdU Flow Cytometry Assay Kits provide flexibility in experimental design, allowing more data to be collected from each sample. Designed for use with either blue, red, or violet laser excitation, these cell proliferation assay kits are fully compatible with R-PE and the sensitive R-PE tandems (Figure 4) as well as with GFP (Figures 5 and 6) and other fluorescent proteins. Because the Click-iT® Plus EdU Flow Cytometry Assay Kits do not require harsh denaturation protocols, R-PE and GFP fluorescence is preserved and antigen recognition sites remain intact, ensuring that multiplex experiments will not be compromised.
Figure 4. Dual-parameter plot of fluorescence from cells labeled with the Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit and PE-Cy®7 anti–Hu CD3 antibody. Jurkat (human T cell leukemia) cells were treated with 10 μM EdU for 2 hr, stained with the PE-Cy®7 conjugate of anti–Hu CD3 antibody, and detected according to the Click-iT® Plus EdU staining protocol. Data were collected and analyzed using an Attune® Acoustic Focusing Cytometer with 488 nm excitation and a 530/30 nm bandpass emission filter for detection of the Alexa Fluor® 488–labeled EdU, and with 488 nm excitation and a 574/26 bandpass emission filter for detection of the PE-Cy®7 anti–Hu CD3 conjugate.
Figure 5. Dual-parameter plot of fluorescence of cells labeled with the Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit and GFP. Erk2-GFP–expressing A375 melanoma cells were treated with 10 μM EdU for 2 hr and detected according to the recommended staining protocol. Data were collected and analyzed using an Attune® Acoustic Focusing Cytometer with 635 nm excitation and a 660/20 nm bandpass emission filter for detection of the Alexa Fluor® 647–labeled EdU, and with 488 nm excitation and a 530/30 bandpass emission filter for detection of GFP.
Figure 6. Dual-parameter plot of fluorescence of cells labeled with the Click-iT® Plus EdU Pacific Blue™ Flow Cytometry Assay Kit and GFP. Erk2-GFP–expressing A375 melanoma cells were treated with 10 μM EdU for 2 hr and detected according to the recommended staining protocol. Data were collected and analyzed using an Attune® Acoustic Focusing Cytometer with 405 nm excitation and a 450/20 nm bandpass emission filter for detection of the Pacific Blue™ dye–labeled EdU, and with 488 nm excitation and a 530/30 bandpass emission filter for detection of GFP.
Even more Click-iT® Plus tools
In addition to the imaging and flow cytometry kits, the Click-iT® Plus technology is available in the Click-iT® Plus Alexa Fluor® Picolyl Azide Toolkits, which contain the reagents needed to perform copper-catalyzed click reactions with copper-sensitive compounds. These toolkits provide Alexa Fluor® picolyl azide and Click-iT® reaction buffers as well as copper sulfate and copper protectant, and they allow you to optimize your own Click–iT® Plus detection of alkyne-containing biomolecules in vitro, in cells, or in tissue samples.