Click-iT™ Plus EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 555 dye (Invitrogen™)

Click-iT Plus EdU Cell Proliferation imaging kits have been optimized for fluorescence microscopy applications and are a superior alternative to traditional proliferation assays. In this assay the modified thymidine analogue EdU (5-ethynyl-2'-deoxyuridine, a nucleoside analog of thymidine) is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor™ dye in a fast, highly-specific, mild click reaction.

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• Simple—works the first time, every time, in less time than traditional methods
• Efficient—no denaturation steps or harsh treatment required
• Content-rich results—improved preservation of cell morphology, antigen structure, GFP fluorescent signal, and DNA integrity
• Consistent—not dependent on variable antibody lots for detection

The kit contains all of the components needed to label and detect the incorporated EdU as well as perform cell cycle analysis on samples from adherent cells. For cell cycle analysis, the kit includes a blue fluorescent Hoechst 33342 dye. The kit contains sufficient reagents for labeling 50 18x18 coverslips using 500 μL of reaction buffer per test.

Avoid the Harsh Treatments Associated with BrdU Method
Measuring changes to cell proliferation is a fundamental method for assessing cell health, determining genotoxicity, and evaluating anti-cancer drugs. The most accurate method of doing this is by directly measuring DNA synthesis. The traditional method utilizes the nucleoside analog BrdU (5-bromo-2'-deoxyuridine, a nucleoside analog of thymidine), which is incorporated into newly transcribed DNA. After incorporation the samples are treated with harsh methods (HCl, heat, or enzymes) to denature the DNA and expose the BrdU molecules to detection by anti-BrdU antibodies. However, the BrdU method to measure cell proliferation is time consuming and difficult to perform consistently. The harsh treatments necessary for this method can adversely affect sample integrity, cell morphology, image quality, and the ability to multiplex.

The Click-iT™ Plus EdU assay measures the rate of new DNA synthesis based on incorporation of the nucleoside analog EdU into DNA. Detection is achieved through a catalyzed 'click' reaction that is completed typically within 30 minutes. The click reaction uses bioorthogonal (biologically unique) moieties to fluorescently label proliferating cells, producing low backgrounds and high detection sensitivities. Because of the mild reaction conditions the Click-iT™ Plus assays can accurately determine cell proliferation while preserving cell morphology, DNA integrity, antigen binding sites, and the fluorescent signal from GFP. Preservation of DNA integrity allows for DNA staining, including staining with dyes used for cell cycle analysis.