Invitrogen™ Click-iT™ EdU Alexa Fluor™ 594 HCS Assay

Mild fixation and detergent permeabilization sufficient for Click-iT EdU detection reagent to gain access to the DNA. The Click-iT EdU Alexa Fluor 594 HCS Assay is a superior alternative to traditional proliferation assays that is optimized for high-content imaging applications. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with a bright, photostable Alexa Fluor dye in a fast, highly specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol. The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently. The harsh treatment can also adversely effect sample integrity and quality, which makes co-staining with other antibodies challenging.

• Simple: works the first time, every time, in less time
• Efficient: no denaturation steps or harsh treatment required
• Content-rich results: better preservation of cell morphology, antigen structure, and DNA integrity
• Consistent: not dependent on variable antibody lots for detection
• Less hazardous

Superior Proliferation Methodology

The Click-iT EdU Alexa Fluor 594 HCS Assay provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT EdU detection reagent to gain access to the DNA. As a consequence, the Click-iT EdU HCS assay is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.

The Click-iTassay can be used on cells in culture or in vivo following administration of EdU by feeding or injection methods.

The Click-iTassay can be used with BrdU in dual pulse experiments by using the ant-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.

The Click-iTtechnology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed-cell labeling.

Cell Analysis, Cell Cycle, Cell Proliferation, Cell Viability, Proliferation and Function, Cellular Imaging, DNA Replication and Repair, High-Content Screening (HCS)