The Click-iT® Plus EdU Alexa Fluor® 488 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 488 nm laser of the flow cytometer. The Click-iT® Plus formulation is compatible with standard fluorophores, including R-PE and R-PE tandems, as well as fluorescent proteins.• Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins• Accurate—superior results compared to BrdU assays• Fast—results in as little as 60 minutesMultiplexableThe Click-iT® Plus formulation provides increased multiplexibility compared to the original Click-iT® EdU Flow Cytometry assays. Click-iT® Plus EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry, without loss of the accuracy or speed of the original Click-iT® EdU assay.An Advanced Method Giving You Results Superior to BrdUThe most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor® dye. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population.Mild Conditions Allow Use with Cell Cycle Dyes and AntibodiesThe small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® Plus detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it can be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution, as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use Click-iT® Plus EdU assay is compatible with cell cycle dyes. The Click-iT® Plus EdU assay can also be multiplexed with antibodies against surface and intracellular markers, as well as conjugates labeled with standard fluorophores including R-PE, R-PE tandems, and fluorescent proteins (GFP and mCherry).Quick and Simple ProtocolThe Click-iT® Plus EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling. However, EdU is compatible with other fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:1. Treat cells with EdU.2. Fix and permeabilize cells.3. Detect S-phase cells with Click-iT® Plus detection cocktail for 30 min.4. Wash once.5. Analyze.Results can be seen in as little as 60 minutes in some circumstances, but we recommend 90 minutes for all applications. |
| 图1.Jurkat cells stained with CD3 antibody-PE-Cy®5.5 conjugateJurkat (human T-cell leukemia) cells were treated with 10 µM EdU for 2 hours, stained with CD3 mouse anti-human mAb PE-Cy®5.5 (Cat. No. MHCD0318) and detected according to the recommended staining protocol. The figures show a clear separation of proliferating cells which have incorporated EdU and nonproliferating cells which have not. Panel A shows data from cells labeled with Alexa Fluor® 488 picolyl azide analyzed on an Attune® Acoustic Focusing Cytometer using 488 nm excitation and a 530/30 nm bandpass emission filter; Panel B shows the same cells using 488 nm excitation and a 574/26 nm bandpass emission filter. The black outlined histogram is the cells stained with CD3 mouse anti-human mAb PE-Cy®5.5 and Click-iT® Plus EdU Alexa Fluor® 488 picolyl azide. The gray outlined histogram is theCD3 mouse anti-human mAb PE-Cy®5.5 positive control cells treated the same but without copper in the reaction.图2.Alexa Fluor® 488 Click-iT® Plus EdU Flow Cytometry Assay Kits, mCherry, and FxCycle™ FarRedA549 cells were transduced with adenovirus for 48 hours, treated with 10 µM EdU for 2 hours and detected according to the recommended staining protocol. The figures show a clear separation of proliferating cells that have incorporated EdU and nonproliferating cells that have not. Panel A shows data from cells labeled with Alexa Fluor® 488 picolyl azide on an LSRII flow cytometer using 488 nm excitation and a 530/30 nm bandpass emission filter. Panel B shows the mCherry-expressing cells clicked with Click-iT® Plus EdU Alexa Fluor® 488 picolyl azide using 532 nm excitation and a 585/42 nm bandpass emission filter to display the mCherry fluorescence. Panel C shows the mCherry-expressing control cells clicked with Click-iT® Plus EdU Alexa Fluor® 488 picolyl azide but without the copper in the reaction, using 532 nm excitation and a 585/42 nm bandpass emission filter to display the positive control mCherry fluorescence. Panel D shows the dual parameter plot of the Click-iT® Plus EdU Alexa Fluor® 488 and FxCycle™ FarRed. Data were collected and analyzed using an LSRII flow cytometer using 488 nm excitation and a 530/30 nm bandpass emission filter for detection of the EdU Alexa Fluor® 488 picolyl azide and 635 nm excitation and a 660/20 bandpass emission filter for detection of the FxCycle Violet™ fluorescence. This figure combines DNA content with EdU; cells that are positive for both labels are in S-phase of the cell cycle. |
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